Methods and materials for treatment of pompe&#39;s disease

ABSTRACT

This document relates to molecular complexes having acid alpha glucosidase activity and at least one modification that results in enhanced ability of the molecular complex to be transported to the interior of a mammalian cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/611,485, filed Mar. 15, 2012. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

TECHNICAL FIELD

This invention relates to isolated molecular complexes having acid alphaglucosidase activity, and more particularly to molecular complexescomprising at least two polypeptides derived by proteolysis from aprecursor molecule, wherein the molecular complex includes at least onemodification that results in enhanced ability of the molecular complexto be transported to the interior of a mammalian cell.

BACKGROUND

Pompe's disease (also referred to as glycogen-storage disease type II oracid-maltase deficiency) is a rare autosomal recessive disorder thatresults in an accumulation of glycogen in the lysosome due to adeficiency of acid alpha glucosidase (GAA). The build-up of glycogencauses progressive muscle weakness (myopathy) throughout the body andaffects various body tissues, including the heart, skeletal muscles,live, and nervous system.

Pompe's disease is broadly classified into infantile and late onsetforms. In the infantile-onset form, infants typically present duringearly infancy (4-8 months of age) with weakness and floppiness, and areunable to hold up their heads and cannot do other motor tasks common fortheir age, such as rolling over. Without treatment, infants with Pompe'sdisease usually die before 12 months of age due to heart failure andrespiratory weakness. See, United Pompe Foundation. Late onset forms(including juvenile and adult forms), have a later onset and progressmore slowly than the infantile form. Recombinant human GAA (Myozyme® orLumizyme®) is used to treat Pompe's disease. However, Myozyme® orLumizyme® are both very expensive, with costs well over $300,000 peryear. As such, there is a need for improved treatments for Pompe'sdisease.

SUMMARY

In one aspect, this document features an isolated molecular complexhaving acid alpha glucosidase (GAA) activity and that includes at leasttwo polypeptides (e.g., at least three or at least four polypeptides),each polypeptide having at least 85% (e.g., at least 90%, 95%, 99%, or100%) sequence identity to a segment of the amino acid sequence setforth in SEQ ID NO: 1, each segment being derived by proteolysis of theamino acid sequence set forth in SEQ ID NO: 1 at one or more sitesbetween amino acid 50 and amino acid 74 (e.g., between amino acid 56 andamino acid 68 or between amino acid 60 and amino acid 65). The molecularcomplex includes at least one modification that results in enhancedability of the molecular complex to be transported to the interior of amammalian cell. Proteolysis of the amino acid sequence set forth in SEQID NO:1 further can include cleavage at one or more sites between aminoacid 719 and amino acid 746 or cleavage at one or more sites betweenamino acid 137 and amino acid 151 of the amino acid sequence set forthin SEQ ID NO:1. Proteolysis further can include cleavage at one or moresites between amino acid 719 and amino acid 746 of the amino acidsequence set forth in SEQ ID NO:1 and cleavage at one or more sitesbetween amino acid 137 and amino acid 151 of the amino acid sequence setforth in SEQ ID NO:1.

In any of the molecular complexes described herein, at least one of thepolypeptides can include one or more phosphorylated N-glycans and themodification can include uncapping and demannosylation of at least onephosphorylated N-glycan. At least 40% (e.g., at least 60%, 80%, 90%,95%, or 99%) of the N-glycans on at least one of the polypeptides can beuncapped and demannosylated.

In any of the molecular complexes described herein, for one of the atleast two polypeptides, the segment includes amino acids 22 to 57 of SEQID NO:1, and wherein for one of the at least two polypeptides, thesegment includes amino acids 66 to 896 of SEQ ID NO:1.

In any of the molecular complexes described herein containing at leastthree polypeptides, for one of the at least three polypeptides, thesegment includes amino acids 22 to 57 of SEQ ID NO:1, wherein for one ofthe at least three polypeptides, the segment includes amino acids 66 to726 of SEQ ID NO:1, and wherein for one of the at least threepolypeptides, the segment includes amino acids 736 to 896 of SEQ IDNO:1.

In any of the molecular complexes described herein containing at leastfour polypeptides, for one of the at least four polypeptides, thesegment includes amino acids 22 to 57 of SEQ ID NO:1, wherein for one ofthe at least four polypeptides, the segment includes amino acids 66 to143 of SEQ ID NO:1, wherein for one of the at least four polypeptides,the segment includes amino acids 158 to 726 of SEQ ID NO:1, and whereinfor one of the at least four polypeptides, the segment includes aminoacids 736 to 896 of SEQ ID NO:1.

In any of the molecular complexes described herein, the at least onemodification can include any one of the following fused to at least onepolypeptide in the molecular complex: a ligand for an extracellularreceptor, a targeting domain that binds an extracellular domain of areceptor on the surface of a target cell, a urokinase-type plasminogenreceptor, or the recognition domain of human insulin-like growth factorII.

This document also features compositions that include any of themolecular complexes described herein, wherein the molecular complex islyophilized. The composition can be packaged as a single use vial.

This document also features a pharmaceutical composition that includesany of the molecular complexes described herein and a pharmaceuticallyacceptable carrier. The composition can be formulated for intravenous orsubcutaneous administration. The composition can be formulated forintravenous infusion.

In another aspect, this document features a method of treating Pompe'sdisease. The method includes administering any of the compositionsdescribed herein to a patient diagnosed with Pompe's disease. Thepatient can be diagnosed with infantile onset Pompe's disease or lateonset Pompe's disease.

This document also features a method for making a molecular complex. Themethod includes contacting a polypeptide having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:1 with aprotease having at least 85% (e.g., at least 90%, at least 95%, at least99%, or 100%) sequence identity to the amino acid sequence set forth inSEQ ID NO:8, wherein the protease cleaves the polypeptide at one or moresites between amino acid 50 and amino acid 74 (e.g., between amino acid56 and amino acid 68 or between amino acid 60 and amino acid 65). Thecontacting step can be performed in vitro.

This document also features a method for making a molecular complex thatincludes uncapped and demannosylated phosphorylated N-glycans. Themethod includes contacting a molecular complex with a mannosidasecapable of (i) hydrolyzing a mannose-1-phospho-6-mannose moiety tomannose-6-phosphate and (ii) hydrolyzing terminal alpha-1,2 mannose,alpha-1,3 mannose and/or alpha-1,6 mannose linkages, the molecularcomplex having GAA activity and including at least two polypeptides,each polypeptide having at least 85% sequence identity to a segment ofthe amino acid sequence set forth in SEQ ID NO: 1, each segment beingderived by proteolysis of the amino acid sequence set forth in SEQ IDNO: 1 at one or more sites between amino acid 50 and amino acid 74(e.g., between amino acid 56 and amino acid 68 or between amino acid 60and amino acid 65), wherein before the contacting, at least one of thepolypeptides includes phosphorylated N-glycans containing one or moremannose-1-phospho-6-mannose moieties. The mannosidase can be a family 38glycosyl hydrolase (e.g., a Canavalia ensiformis mannosidase or aYarrowia lipolytica mannosidase). The contacting can occur in arecombinant fungal cell expressing the mannosidase.

This document also features a method of making a molecular complex thatincludes uncapped and demannosylated phosphorylated N-glycans. Themethod includes contacting a molecular complex with a mannosidasecapable of hydrolyzing terminal alpha-1,2 mannose, alpha-1,3 mannoseand/or alpha-1,6 mannose linkages, the molecular complex having GAAactivity and comprising at least two polypeptides, each polypeptidehaving at least 85% sequence identity to a segment of the amino acidsequence set forth in SEQ ID NO: 1, each segment being derived byproteolysis of the amino acid sequence set forth in SEQ ID NO: 1 at oneor more sites between amino acid 50 and amino acid 74 (e.g., betweenamino acid 56 and amino acid 68 or between amino acid 60 and amino acid65), wherein at least one of the polypeptides includes prior tocontacting, phosphorylated N-glycans comprising uncappedmannose-6-phosphate moieties. The mannosidase can be a family 47glycosyl hydrolase (e.g., an Aspergillus satoi mannosidase), a family 92glycosyl hydrolase (e.g., a Cellulosimicrobium cellulans mannosidase),or a family 38 glycosyl hydrolase (e.g., a Canavalia ensiformismannosidase). The contacting can occur in a recombinant fungal cellexpressing the mannosidase.

This document also features a method of making a molecular complex thatincludes uncapped and demannosylated phosphorylated N-glycans. Themethod includes contacting a molecular complex with a mannosidasecapable of hydrolyzing a mannose-1-phospho-6-mannose moiety tomannose-6-phosphate, the molecular complex having GAA activity andincluding at least two polypeptides, each polypeptide having at least85% sequence identity to a segment of the amino acid sequence set forthin SEQ ID NO: 1, each segment being derived by proteolysis of the aminoacid sequence set forth in SEQ ID NO: 1 at one or more sites betweenamino acid 50 and amino acid 74 (e.g., between amino acid 56 and aminoacid 68 or between amino acid 60 and amino acid 65), wherein at leastone of the polypeptides includes, before the contacting, one or moremannose-1-phospho-6-mannose moieties. The mannosidase can be a family 38glycosyl hydrolase (e.g., a Canavalia ensiformis mannosidase or aYarrowia lipolytica mannosidase).

In another aspect, this document features a method of making a molecularcomplex that includes uncapped and demannosylated phosphorylatedN-glycans. The method includes a) contacting a molecular complex with amannosidase capable of hydrolyzing a mannose-1-phospho-6-mannose moietyto mannose-6-phosphate to uncap mannose-6-phosphate moieties on at leastone polypeptide in the molecular complex, the molecular complex havingGAA activity and comprising at least two polypeptides, each polypeptidehaving at least 85% sequence identity to a segment of the amino acidsequence set forth in SEQ ID NO: 1, each segment being derived byproteolysis of the amino acid sequence set forth in SEQ ID NO: 1 at oneor more sites between amino acid 50 and amino acid 74 (e.g., betweenamino acid 56 and amino acid 68 or between amino acid 60 and amino acid65); and b) contacting the molecular complex with a mannosidase capableof hydrolyzing terminal alpha-1,2 mannose, alpha-1,3 mannose and/oralpha-1,6 mannose linkages. Step (a) and step (b) can be catalyzed bytwo different enzymes or catalyzed by a single enzyme. The contactingsteps can be performed together or separately, and in either order. Thecontacting can occur in a recombinant fungal host cell, the fungal hostcell expressing a mannosidase capable of catalyzing step (a) and amannosidase capable of catalyzing step (b). The contacting can occur ina recombinant fungal host cell, the fungal host expressing a mannosidasecapable of catalyzing steps (a) and (b).

Any of the molecular complexes described herein that include at leastone uncapped and demannosylated N-glycan can be used to contact amammalian cell, wherein, after the contacting, the molecular complex istransported to the interior of the mammalian cell with enhancedefficiency. The mammalian cell can be a human cell.

This document also features a method of transporting a molecular complexhaving GAA activity to the interior of a cell. The method includescontacting a mammalian cell with the molecular complex, the molecularcomplex including at least two polypeptides, each polypeptide having atleast 85% sequence identity to a segment of the amino acid sequence setforth in SEQ ID NO: 1, each segment being derived by proteolysis of theamino acid sequence set forth in SEQ ID NO: 1 at one or more sitesbetween amino acid 50 and amino acid 74 (e.g., between amino acid 56 andamino acid 68 or between amino acid 60 and amino acid 65); whereinphosphorylated N-glycans on at least one of the polypeptides have beenuncapped and demannosylated as set forth in the methods describedherein. The mammalian cell can be in vitro or in a mammalian subject.The mammalian cell can be a human cell.

In another aspect, this document features a method of transporting amolecular complex having GAA activity to the interior of a cell. Themethod includes contacting a mammalian cell with the molecular complexthat includes at least two polypeptides, each polypeptide having atleast 85% sequence identity to a segment of the amino acid sequence setforth in SEQ ID NO: 1, each segment being derived by proteolysis of theamino acid sequence set forth in SEQ ID NO: 1 at one or more sitesbetween amino acid 50 and amino acid 74 (e.g., between amino acid 56 andamino acid 68 or between amino acid 60 and amino acid 65), the molecularcomplex comprising at least one modification that results in enhancedability of the molecular complex to be transported to the interior of amammalian cell. The mammalian cell can be in vitro or in a mammaliansubject. The mammalian cell can be a human cell. The modification caninclude any one of the following fused to at least one polypeptide inthe molecular complex: a ligand for an extracellular receptor, atargeting domain that binds an extracellular domain of a receptor on thesurface of a target cell, a urokinase-type plasminogen receptor, or therecognition domain of human insulin-like growth factor II.

In another aspect, this document features an isolated fungal cell thatincludes an exogenous nucleic acid encoding an alkaline protease havingat least 85% sequence identity to the amino acid sequence set forth inSEQ ID NO:8.

This document also features an isolated fungal cell comprising a nucleicacid encoding the GAA amino acid sequence set forth in SEQ ID NO:1 and anucleic acid encoding an alkaline protease having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8. The fungalcell produces a molecular complex having GAA activity and comprising atleast two polypeptides, each polypeptide having at least 85% sequenceidentity to a segment of the amino acid sequence set forth in SEQ ID NO:1, each segment being derived by proteolysis of the amino acid sequenceset forth in SEQ ID NO: 1 at one or more sites between amino acid 50 andamino acid 74 (e.g., between amino acid 56 and amino acid 68 or betweenamino acid 60 and amino acid 65) by the alkaline protease. In someembodiments, the fungal cell further comprises a nucleic acid encoding amannosidase, the mannosidase being capable of hydrolyzing amannose-1-phospho-6-mannose moiety to mannose-6-phosphate. In someembodiments, the fungal cell further includes a nucleic acid encoding amannosidase, the mannosidase being capable of hydrolyzing a terminalalpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkage.In some embodiments, the fungal cell further can include a nucleic acidencoding a mannosidase, the mannosidase being capable of (i) hydrolyzinga mannose-1-phospho-6-mannose moiety to mannose-6-phosphate and (ii)hydrolyzing a terminal alpha-1,2 mannose, alpha-1,3 mannose and/oralpha-1,6 mannose linkage. Any of such fungal cells further can includea nucleic acid encoding a polypeptide capable of promoting mannosylphosphorylation and/or be genetically engineered to be deficient in OCH1activity.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, Genbank® Accession Nos, and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present application, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a depiction of the amino acid sequence (SEQ ID NO:1) of humanacid alpha glucosidase (GAA) after cleavage of the signal sequence.

FIG. 2A is a depiction of the nucleotide sequence of the open readingframe (ORF) of DsbA-Cellulosimicrobium cellulans mannosidase 5 (CcMan5)(SEQ ID NO:2).

FIG. 2B is a depiction of the amino acid sequence of the CcMan5polypeptide with the signal sequence in bold (SEQ ID NO: 3).

FIG. 2C is a depiction of the amino acid sequence of the CcMan5polypeptide without signal sequence (SEQ ID NO:4). The predictedmolecular weight of the CcMan5 polypeptide without the signal sequenceis 173 kDa.

FIGS. 3A and 3B are a series of electropherograms depicting the N-glycananalysis of rhGAA treated with CcMan5 and JbMan. Analysis was performedusing DNA sequencer-assisted, fluorophore-assisted carbohydrateelectrophoresis (DSA-FACE). The Y-axis represents the relativefluorescence units as an indication of the amount of each N-glycanstructure. The X-axis represents the relative mobility of each N-glycanstructure through a capillary. In both FIG. 3A and FIG. 3B, panel A is areference sample containing the N-glycans released from RNaseB withPNGaseF. In FIG. 3A, panels B and C contain the N-glycan analysis fromhuGAA (76 kD variant) before and after treatment, respectively, withCcMan5 and JbMan. In FIG. 3B, panels B, C, and D contain the N-glycananalysis from huGAA 76 kD form, 95 kD form, and 110 kD form,respectively.

FIG. 4 is a line graph of the amount of glucose formed per minute withMyozyme (•), 76 kDa GAA (▴), 95 kDa GAA (▾), and 110 kDa GAA (♦) usingrabbit liver glycogen as substrate.

FIG. 5A contains two depictions of the glycogen levels (μg/mg protein)of individual mice in heart. FIG. 5B contains two depictions of theglycogen levels (μg/mg protein) of individual mice in skeletal muscle.Red dots are females, black dots are males. Line represents the medianof each group.

FIG. 6 contains a depiction of the amino acid sequence of the Yarrowialipolytica AMS1 mannosidase (SEQ ID NO: 5).

FIG. 7 contains a depiction of the amino acid sequence of theAspergillus satoi mannosidase (SEQ ID NO:6).

FIG. 8 contains a depiction of the amino acid sequence of theCellulosimicrobium cellulans mannosidase 4 (CcMan4, SEQ ID NO:7), withsignal sequence in bold. The predicted molecular weight of the CcMan4polypeptide without the signal sequence is 184 kDa.

FIG. 9 contains a depiction of the amino acid sequence of theAspergillus oryzae alkaline protease including the signal peptide (21amino acids), pro-peptide (100 amino acids) and mature protein (282amino acids) (SEQ ID NO:9).

FIG. 10 contains a depiction of the nucleotide sequence of the fusionconstruct containing the Y. lipolytica codon optimized sequence encodingthe A. oryzae alkaline protease (SEQ ID NO:10). Restriction sites usedfor cloning are underlined. The nucleotide sequence encoding the linkeris in bold and the nucleotide sequence encoding the His tag (10 Hisresidues) is italicized.

DETAILED DESCRIPTION

In general, this document provides isolated molecular complexes havingacid alpha-glucosidase (GAA) activity and at least one modification thatresults in an enhanced ability to be transported to the interior of amammalian cell. GAA is synthesized as a 110 kDa precursor containingN-linked glycans. The precursor is proteolytically processed to removethe signal sequence and then further proteolytically processed to majorspecies of 95 kDa, 76 kDa, and 70 kDa. However, at least some of thepeptides that are released from the precursor remain associated with themajor species. See, for example, Moreland et al., J. Biol. Chem.,280:6780-6791 (2005). Thus, the molecular complexes having GAA activitydescribed herein include at least two polypeptides (at least two, three,or four polypeptides) that are derived from proteolytic cleavage of theprecursor molecule at one or more sites. At least two polypeptides inthe molecular complex result from proteolytic cleavage at one or moresites in the precursor. For example, proteolysis of the amino acidsequence set forth in SEQ ID NO:1 can be between amino acid 50 and aminoacid 74, e.g., between amino acid 56 and amino acid 68 or between aminoacid 60 and amino acid 65, to produce at least two polypeptides. Amolecular complex containing two polypeptides is referred to as the 95kDa form herein.

In some embodiments, at least three polypeptides in the molecularcomplex result from proteolytic cleavage at two or more sites in theprecursor. For example, proteolysis of the amino acid sequence set forthin SEQ ID NO:1 can include, in addition to cleavage between amino acid50 and amino acid 74 (e.g., between amino acid 56 and amino acid 68 orbetween amino acid 60 and amino acid 65), cleavage at one or more sitesbetween amino acid 719 and amino acid 746 or cleavage at one or moresites between amino acid 137 and amino acid 151 of the amino acidsequence set forth in SEQ ID NO:1. A molecular complex containing threepolypeptides is referred to as the 76 kDa form herein.

In some embodiments, at least four polypeptides in the molecular complexresult from proteolytic cleavage at three or more sites in theprecursor. For example, proteolysis of the amino acid sequence set forthin SEQ ID NO:1 can include, in addition to the cleavage between aminoacid 50 and amino acid 74 (e.g., between amino acid 56 and amino acid 68or between amino acid 60 and amino acid 65), cleavage at one or moresites between amino acid 719 and amino acid 746 of the amino acidsequence set forth in SEQ ID NO:1 and cleavage at one or more sitesbetween amino acid 137 and amino acid 151 of the amino acid sequence setforth in SEQ ID NO:1. A molecular complex containing four polypeptidesis referred to as the 70 kDa form herein.

It will be appreciated that cleavage can occur at one or more sites inone molecule, and that the site of cleavage can be different indifferent molecules.

A commercially available protease mix containing proteases fromAspergillus oryzae (e.g., from Sigma or NovozymesCorp) can be used tocleave the amino acid sequence set forth in SEQ ID NO:1 between aminoacids 50 and 74, e.g., between amino acids 56 and 68 or between aminoacids 60 and 65. Alternatively, an alkaline protease having at least 85%(e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity tothe alkaline protease from Aspergillus oryzae (SEQ ID NO:8) can be used.For example, as described herein, a GAA polypeptide having the aminoacid sequence set forth in SEQ ID NO:1 can be contacted with a proteasehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:8 or SEQ ID NO: 9. SEQ ID NO: 8 is the amino acidsequence of the mature Aspergillus oryzae alkaline protease. SEQ ID NO:9 is the amino acid sequence of the Aspergillus oryzae proteaseincluding the signal peptide, pro-peptide, and mature protein. Thecontacting can occur in vitro using protease that has been isolated fromAspergillus oryzae or that has been recombinantly produced.Alternatively, a fungal host can be engineered such that the GAApolypeptide and alkaline protease are both secreted into the culturemedium, where the alkaline protease can cleave the amino acid sequenceset forth in SEQ ID NO:1 between amino acid 50 and amino acid 74 (e.g.,between amino acids 56 and 68 or between amino acids 60 and 65).

The isolated molecular complexes described herein have at least onemodification that results in an enhanced ability to be transported tothe interior of a mammalian cell. Non-limiting examples of modificationsthat enhance the ability of the complex of being transported to theinterior of a mammalian cell include uncapping and demannosylation ofphosphorylated N-glycans or peptide tags that facilitate transport.Methods and materials are described herein for preparing molecularcomplexes containing tags or uncapped and demannosylated N-glycans.

The isolated molecular complexes described herein are particularlyuseful for treating patients with Pompe disease, including a patientdiagnosed with Pompe's disease, both infantile onset Pompe's disease andlate onset Pompe's disease. Pompe's disease results in an accumulationof glycogen in the lysosome due to a deficiency of GAA. The build-up ofglycogen causes progressive muscle weakness (myopathy) throughout thebody and affects various body tissues, including the heart, skeletalmuscles, live, and nervous system.

Each of the polypeptide in the molecular complex have at least 85%sequence identity (e.g., at least 90%, 95%, 97%, 98%, 99%, or 100%) to asegment of the amino acid sequence set forth in SEQ ID NO: 1, eachsegment being derived by proteolysis of the amino acid sequence setforth in SEQ ID NO: 1 at one or more sites between amino acid 50 andamino acid 74 (e.g., between amino acid 56 and amino acid 68 or betweenamino acid 60 and amino acid 65). The percent identity between aparticular amino acid sequence and the amino acid sequence set forth inSEQ ID NO: 1 can be determined as follows. First, the amino acidsequences are aligned using the BLAST 2 Sequences (B12seq) program fromthe stand-alone version of BLASTZ containing BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained from Fish & Richardson'sweb site (e.g., www.fr.com/blast/) or the U.S. government's NationalCenter for Biotechnology Information web site (www.ncbi.nlm.nih.gov).Instructions explaining how to use the Bl2seq program can be found inthe readme file accompanying BLASTZ. Bl2seq performs a comparisonbetween two amino acid sequences using the BLASTP algorithm. To comparetwo amino acid sequences, the options of Bl2seq are set as follows: —iis set to a file containing the first amino acid sequence to be compared(e.g., C:\seq1.txt); —j is set to a file containing the second aminoacid sequence to be compared (e.g., C:\seq2.txt); —p is set to blastp;—o is set to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\B12seq—i c:\seq1.txt—jc:\seq2.txt—p blastp—o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences. Similar procedures can be following for nucleic acidsequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the full-length polypeptide amino acid sequencefollowed by multiplying the resulting value by 100.

It is noted that the percent identity value is rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenGAA polypeptide can be modified such that optimal expression in aparticular species (e.g., bacteria or fungus) is obtained, usingappropriate codon bias tables for that species.

In one embodiment, a molecular complex can include at least twopolypeptides, where one of the polypeptides includes amino acids 22 to57 of SEQ ID NO:1, and another polypeptide includes amino acids 66 to896 of SEQ ID NO:1.

In one embodiment, a molecular complex can include at least threepolypeptides, wherein one of the polypeptides includes amino acids 22 to57 of SEQ ID NO:1, one polypeptide includes amino acids 66 to 726 of SEQID NO:1, and one polypeptide includes amino acids 736 to 896 of SEQ IDNO:1.

In one embodiment, a molecular complex can include at least fourpolypeptides, wherein one of the polypeptides includes amino acids 22 to57 of SEQ ID NO:1, one polypeptide includes amino acids 66 to 143 of SEQID NO:1, one polypeptide includes amino acids 158 to 726 of SEQ ID NO:1,and one polypeptide includes amino acids 736 to 896 of SEQ ID NO:1.

Biologically active variants of GAA can contain additions, deletions, orsubstitutions relative to the sequences set forth in SEQ ID NO: 1. GAAproteins with substitutions will generally have not more than 10 (e.g.,not more than one, two, three, four, five, six, seven, eight, nine, orten) conservative amino acid substitutions. A conservative substitutionis the substitution of one amino acid for another with similarcharacteristics. Conservative substitutions include substitutions withinthe following groups: valine, alanine and glycine; leucine, valine, andisoleucine; aspartic acid and glutamic acid; asparagine and glutamine;serine, cysteine, and threonine; lysine and arginine; and phenylalanineand tyrosine. The non-polar hydrophobic amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Any substitution of one member of the above-mentionedpolar, basic or acidic groups by another member of the same group can bedeemed a conservative substitution. By contrast, a non-conservativesubstitution is a substitution of one amino acid for another withdissimilar characteristics.

In some embodiments, a GAA polypeptide can be a fusion protein with aheterologous amino acid sequence such as a sequence used forpurification of the recombinant protein (e.g., FLAG, polyhistidine(e.g., hexahistidine), hemagluttanin (HA), glutathione-S-transferase(GST), or maltose-binding protein (MBP)).

In some embodiments, the heterologous amino acid sequence is used toenhance the efficiency of transport of the molecular complex into amammalian cell. For example, at least one of the polypeptides in acomplex can be fused to a ligand for an extracellular receptor, atargeting domain that binds an extracellular domain of a receptor on thesurface of a target cell, a urokinase-type plasminogen receptor, ordomains of human insulin-like growth factor II that bind to themannose-6-phosphate receptor (e.g., amino acids 1-67 or 1-87; at leastamino acids 48-55; at least amino acids 8-28 and 41-61; or at leastamino acids 8-87 of human insulin-like growth factor; a sequence variantthereof of human insulin-like growth factor II (e.g., R68A) or truncatedform of human insulin-like growth factor (e.g., C-terminally truncatedfrom position 62)). The heterologous amino acid sequence can be fused atthe N-terminus or C-terminus of the polypeptide. In one embodiment, apeptide tag is fused to the N- or C-terminus of the polypeptide by aspacer (e.g., 5-30 amino acids or 10-25 amino acids). See, for example,U.S. Pat. No. 7,785,856.

Heterologous amino sequences also can be proteins useful as diagnosticor detectable markers, for example, luciferase, green fluorescentprotein (GFP), or chloramphenicol acetyl transferase (CAT).

In certain host cells (e.g., yeast host cells), expression and/orsecretion of the target protein can be increased through use of aheterologous signal sequence. In some embodiments, the fusion proteincan contain a carrier (e.g., KLH) useful, e.g., in eliciting an immuneresponse for antibody generation) or endoplasmic reticulum or Golgiapparatus retention signals. Heterologous sequences can be of varyinglength and in some cases can be a longer sequences than the full-lengthtarget proteins to which the heterologous sequences are attached.

Methods of Demannosylating, or Uncapping and DemannosylatingGlycoproteins

Glycoproteins containing a phosphorylated N-glycan can bedemannosylated, and glycoproteins containing a phosphorylated N-glycancontaining a mannose-1-phospho-6-mannose linkage or moiety can beuncapped and demannosylated by contacting the glycoprotein with amannosidase capable of (i) hydrolyzing a mannose-1-phospho-6-mannoselinkage or moiety to mannose-6-phosphate and (ii) hydrolyzing a terminalalpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkage ormoiety. Non-limiting examples of such mannosidases include a Canavaliaensiformis (Jack bean) mannosidase and a Yarrowia lipolytica mannosidase(e.g., AMS1). Both the Jack bean and AMS1 mannosidase are family 38glycoside hydrolases.

The Jack bean mannosidase is commercially available, for example, fromSigma-Aldrich (St. Louis, Mo.) as an ammonium sulfate suspension(Catalog No. M7257) and a proteomics grade preparation (Catalog No.M5573). Such commercial preparations can be further purified, forexample, by gel filtration chromatography to remove contaminants such asphosphatases. The Jack bean mannosidase contains a segment with thefollowing amino acid sequence NKIPRAGWQIDPFGHSAVQG (SEQ ID NO: 11). SeeHoward et al., J. Biol. Chem., 273(4):2067-2072, 1998.

The Yarrowia lipolytica AMS1 mannosidase can be recombinantly produced.The amino acid sequence of the AMS1 polypeptide is set forth in SEQ IDNO:5 (see also FIG. 6).

In some embodiments, the uncapping and demannosylating steps arecatalyzed by two different enzymes. For example, uncapping of amannose-1-phospho-6 mannose linkage or moiety can be performed using amannosidase from Cellulosimicrobium cellulans (e.g., CcMan5). Thenucleotide sequence encoding the CcMan5 polypeptide is set forth in SEQID NO:2 (see FIG. 2A). The amino acid sequence of the CcMan5 polypeptidecontaining signal sequence is set forth in SEQ ID NO: 3 (see FIG. 2B).The amino acid sequence of the CcMan5 polypeptide without signalsequence is set forth in SEQ ID NO:4 (see FIG. 2C). In some embodiments,a biologically active fragment of the CcMan5 polypeptide is used. Forexample, a biologically active fragment can includes residues 1-774 ofthe amino acid sequence set forth in SEQ ID NO:4. See also WO2011/039634. The CcMan5 mannosidase is a family 92 glycoside hydrolase.

Demannosylation of an uncapped glycoprotein or molecular complexes ofglycoproteins can be catalyzed using a mannosidase from Aspergillussatoi (As) (also known as Aspergillus phoenicis) or a mannosidase fromCellulosimicrobium cellulans (e.g., CcMan4). The Aspergillus satoimannosidase is a family 47 glycoside hydrolase and the CcMan4mannosidase is a family 92 glycoside hydrolase. The amino acid sequenceof the Aspergillus satoi mannosidase is set forth in FIG. 7 (SEQ IDNO:6) and in GenBank Accession No. BAA08634. The amino acid sequence ofthe CcMan4 polypeptide is set forth in FIG. 8 (SEQ ID NO:7).

Demannosylation of an uncapped glycoprotein or molecular complexes ofglycoproteins also can be catalyzed using a mannosidase from the family38 glycoside hydrolases such as a Canavalia ensiformis (Jack bean)mannosidase or a Yarrowia lipolytica mannosidase (e.g., AMS1). Forexample, CcMan5 can be used to uncap a mannose-1-phospho-6 mannosemoiety on a glycoprotein (or molecular complex of glycoproteins) and theJack bean mannosidase can be used to demannosylate the uncappedglycoprotein (or molecular complex of glycoproteins).

To produce demannosylated glycoproteins (or molecular complexes ofglycoproteins), or uncapped and demannosylated glycoproteins (ormolecular complexes of glycoproteins), a target molecule (or molecularcomplex) containing a mannose-1-phospho-6 mannose linkage or moiety iscontacted under suitable conditions with a suitable mannosidase(s)and/or a cell lysate containing a suitable recombinantly producedmannosidase(s). Suitable mannosidases are described above. The celllysate can be from any genetically engineered cell, including a fungalcell, a plant cell, or animal cell. Non-limiting examples of animalcells include nematode, insect, plant, bird, reptile, and mammals suchas a mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow,horse, whale, monkey, or human.

Upon contacting the target molecule (e.g., molecular complex) with thepurified mannosidases and/or cell lysate, themannose-1-phospho-6-mannose linkage or moiety can be hydrolyzed tophospho-6-mannose and the terminal alpha-1,2 mannose, alpha-1,3 mannoseand/or alpha-1,6 mannose linkage or moiety of such a phosphatecontaining glycan can be hydrolyzed to produces an uncapped anddemannosylated target molecule. In some embodiments, one mannosidase isused that catalyzes both the uncapping and demannosylating steps. Insome embodiments, one mannosidase is used to catalyze the uncapping stepand a different mannosidase is used to catalyze the demannosylatingstep. Following processing by the mannosidase, the target molecule ormolecular complex can be isolated.

Suitable methods for obtaining cell lysates that preserve the activityor integrity of the mannosidase activity in the lysate can include theuse of appropriate buffers and/or inhibitors, including nuclease,protease and phosphatase inhibitors that preserve or minimize changes inN-glycosylation activities in the cell lysate. Such inhibitors include,for example, chelators such as ethylenediamine tetraacetic acid (EDTA),ethylene glycol bis(P-aminoethyl ether) N,N,N1,N1-tetraacetic acid(EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, leupeptin, antipain and the like, and phosphataseinhibitors such as phosphate, sodium fluoride, vanadate and the like.Appropriate buffers and conditions for obtaining lysates containingenzymatic activities are described in, e.g., Ausubel et al. CurrentProtocols in Molecular Biology (Supplement 47), John Wiley & Sons, NewYork (1999); Harlow and Lane, Antibodies: A Laboratory Manual ColdSpring Harbor Laboratory Press (1988); Harlow and Lane, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); TietzTextbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. W.B.Saunders, Philadelphia, (1999).

A cell lysate can be further processed to eliminate or minimize thepresence of interfering substances, as appropriate. If desired, a celllysate can be fractionated by a variety of methods well known to thoseskilled in the art, including subcellular fractionation, andchromatographic techniques such as ion exchange, hydrophobic and reversephase, size exclusion, affinity, hydrophobic charge-inductionchromatography, and the like.

In some embodiments, a cell lysate can be prepared in which wholecellular organelles remain intact and/or functional. For example, alysate can contain one or more of intact rough endoplasmic reticulum,intact smooth endoplasmic reticulum, or intact Golgi apparatus. Suitablemethods for preparing lysates containing intact cellular organelles andtesting for the functionality of the organelles are described in, e.g.,Moreau et al. (1991) J. Biol. Chem. 266(7):4329-4333; Moreau et al.(1991) J. Biol. Chem. 266(7):4322-4328; Rexach et al. (1991) J. CellBiol. 114(2):219-229; and Paulik et al. (1999) Arch. Biochem. Biophys.367(2):265-273.

Upon contact of a mammalian cell with a molecular complex containinguncapped and demannosylated phosphorylated N-glycans, the molecularcomplex can be transported to the interior of the mammalian cell (e.g.,a human cell). A molecular complex having an uncapped, but notdemannosylated, phosphorylated N-glycan does not substantially bindmannose-6-phosphate receptors on mammalian cells, and as such, is notefficiently transported to the interior of the cell. As used herein,“does not substantially bind” means that less than 15% (e.g., less than14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, or less, or 0%) of theglycoprotein molecules bind to mannose-6-phosphate receptors onmammalian cells. However, if such a molecular complex is contacted witha mannosidase capable of hydrolyzing a terminal alpha-1,2 mannoselinkage or moiety when the underlying mannose is phosphorylated, ademannosylated glycoprotein is produced that substantially binds to themannose-6-phosphate receptor on the mammalian cells and is efficientlytransported to the interior of the cell. As used herein “substantiallybinds” means that 15% or more (e.g., greater than 16%, 18%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%)of the molecular complex binds to mannose-6-phosphate receptors onmammalian cells. It is understood that a preparation (e.g., arecombinant host cell or a cell-free preparation) containing an enzymethat uncaps but does not demannosylate phosphorylated N-glycans could becontaminated with an enzyme that demannosylates phosphorylatedN-glycans. A target protein sample after contact with such a preparationcan contain protein molecules with some phosphorylated N-glycans thatare uncapped only and others that are uncapped and demannosylated.Naturally those protein molecules containing uncapped and demannosylatedphosphorylated N-glycans can substantially bind to mannose-6-phosphatereceptors. The above definition of “does not substantially bind” doesnot apply to such a target protein sample since the phosphorylatedN-glycans on the protein molecules cannot be characterized as uncappedbut not demannosylated.

Thus, this document provides methods of converting a molecular complexfrom a first form that does not bind to a mannose-6-phosphate receptoron a mammalian cell to a second form that does bind to amannose-6-phosphate receptor on a mammalian cell. In the first form, themolecular complex in which at least one of the polypeptides in thecomplex comprises one or more N-glycans containing one or more mannoseresidues that are linked at the 1 position to a mannose residue thatcontains a phosphate residue at the 6 position. In such methods, thefirst form of the molecular complex is contacted with a mannosidase thatdemannosylates the terminal mannose residues to result in the mannosecontaining the phosphate at the 6 position to become the terminalmannose. In some embodiments, the mannosidase has both uncapping anddemannosylating activity (e.g., Canavalia ensiformis (Jack bean) orYarrowia lipolytica AMS1 mannosidase). In some embodiments, themannosidase does not have uncapping activity (e.g., a mannosidase fromAspergillus satoi or a mannosidase from Cellulosimicrobium cellulans(e.g., CcMan4)).

Transport of a glycoprotein or molecular complex to the interior of thecell can be assessed using a cell uptake assay. For example, mammaliancells and a molecular complex containing uncapped and demannosylatedphosphorylated N-glycans can be incubated, then the cells washed andlysed. Cell lysates can be assessed for the presence of the GAA complex(e.g., by Western blotting) or by activity of GAA in the cell lysate.For example, uptake can be assessed in fibroblasts deficient in acidalpha glucosidase activity. Intracellular activity of alpha glucosidasecan be assessed using the 4-methylumbelliferyl-alpha-D-glucopyranoside(4-MUG) assay. Cleavage of the substrate 4-MUG by a glucosidase leads tothe generation of the fluorigenic product 4-MU, which can be visualizedor detected by irradiation with UV light.

Recombinant Production of Polypeptides

Isolated nucleic acid molecules encoding polypeptides (e.g., amannosidase, an alkaline protease, or GAA or a fragment thereof) can beproduced by standard techniques. The terms “nucleic acid” and“polynucleotide” are used interchangeably herein, and refer to both RNAand DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA)containing nucleic acid analogs. Polynucleotides can have anythree-dimensional structure. A nucleic acid can be double-stranded orsingle-stranded (i.e., a sense strand or an antisense strand).Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers, as wellas nucleic acid analogs.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that are present in anaturally-occurring genome, including nucleic acids that normally flankone or both sides of the nucleic acid in a naturally-occurring genome(e.g., a yeast genome). The term “isolated” as used herein with respectto nucleic acids also includes any non-naturally-occurring nucleic acidsequence, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in anaturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., any paramyxovirus,retrovirus, lentivirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not considered an isolated nucleicacid.

The term “exogenous” as used herein with reference to nucleic acid and aparticular host cell refers to any nucleic acid that does not occur in(and cannot be obtained from) that particular cell as found in nature.Thus, a non-naturally-occurring nucleic acid is considered to beexogenous to a host cell once introduced into the host cell. It isimportant to note that non-naturally-occurring nucleic acids can containnucleic acid subsequences or fragments of nucleic acid sequences thatare found in nature provided that the nucleic acid as a whole does notexist in nature. For example, a nucleic acid molecule containing agenomic DNA sequence within an expression vector isnon-naturally-occurring nucleic acid, and thus is exogenous to a hostcell once introduced into the host cell, since that nucleic acidmolecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of yeast x is an exogenousnucleic acid with respect to a cell of yeast y once that chromosome isintroduced into a cell of yeast y.

Polymerase chain reaction (PCR) techniques can be used to obtain anisolated nucleic acid containing a nucleotide sequence described herein.PCR can be used to amplify specific sequences from DNA as well as RNA,including sequences from total genomic DNA or total cellular RNA.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of the template tobe amplified. Various PCR strategies also are available by whichsite-specific nucleotide sequence modifications can be introduced into atemplate nucleic acid. Isolated nucleic acids also can be chemicallysynthesized, either as a single nucleic acid molecule (e.g., usingautomated DNA synthesis in the 3′ to 5′ direction using phosphoramiditetechnology) or as a series of oligonucleotides. For example, one or morepairs of long oligonucleotides (e.g., >100 nucleotides) can besynthesized that contain the desired sequence, with each pair containinga short segment of complementarity (e.g., about 15 nucleotides) suchthat a duplex is formed when the oligonucleotide pair is annealed. DNApolymerase is used to extend the oligonucleotides, resulting in asingle, double-stranded nucleic acid molecule per oligonucleotide pair,which then can be ligated into a vector. Isolated nucleic acids also canbe obtained by mutagenesis of, e.g., a naturally occurring DNA.

To recombinantly produce a polypeptide (e.g., a mannosidase, an alkalineprotease, or GAA or fragment thereof), a vector is used that contains apromoter operably linked to nucleic acid encoding the polypeptide. Asused herein, a “promoter” refers to a DNA sequence that enables a geneto be transcribed. The promoter is recognized by RNA polymerase, whichthen initiates transcription. Thus, a promoter contains a DNA sequencethat is either bound directly by, or is involved in the recruitment, ofRNA polymerase. A promoter sequence can also include “enhancer regions,”which are one or more regions of DNA that can be bound with proteins(namely, the trans-acting factors, much like a set of transcriptionfactors) to enhance transcription levels of genes (hence the name) in agene-cluster. The enhancer, while typically at the 5′ end of a codingregion, can also be separate from a promoter sequence and can be, e.g.,within an intronic region of a gene or 3′ to the coding region of thegene.

As used herein, “operably linked” means incorporated into a geneticconstruct (e.g., vector) so that expression control sequenceseffectively control expression of a coding sequence of interest.

Expression vectors can be introduced into host cells (e.g., bytransformation or transfection) for expression of the encodedpolypeptide, which then can be purified. Expression systems that can beused for small or large scale production of polypeptides (e.g., amannosidase, alkaline protease, or GAA or fragment thereof) include,without limitation, microorganisms such as bacteria (e.g., E. coli)transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vectors containing the nucleic acid molecules, and fungal(e.g., Yarrowia lipolytica, Arxula adeninivorans, Pichia pastoris,Hansenula polymorphs, Ogataea minuta, Pichia methanolica, Aspergillusniger, Trichoderma reesei, and Saccharomyces cerevisiae) transformedwith recombinant fungal expression vectors containing the nucleic acidmolecules. Useful expression systems also include insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the nucleic acid molecules, and plant cell systems infectedwith recombinant virus expression vectors (e.g., tobacco mosaic virus)or transformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the nucleic acid molecules. Mannosidase or alkalineprotease polypeptides also can be produced using mammalian expressionsystems, which include cells (e.g., immortalized cell lines such as COScells, Chinese hamster ovary cells, HeLa cells, human embryonic kidney293 cells, and 3T3 L1 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,the metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter and the cytomegalovirus promoter).

Recombinant polypeptides such as a mannosidase can be tagged with aheterologous amino acid sequence such FLAG, polyhistidine (e.g.,hexahistidine), hemagluttanin (HA), glutathione-S-transferase (GST), ormaltose-binding protein (MBP) to aid in purifying the protein. Othermethods for purifying proteins include chromatographic techniques suchas ion exchange, hydrophobic and reverse phase, size exclusion,affinity, hydrophobic charge-induction chromatography, and the like(see, e.g., Scopes, Protein Purification: Principles and Practice, thirdedition, Springer-Verlag, New York (1993); Burton and Harding, J.Chromatogr. A 814:71-81 (1998)).

In Vivo Methods of Uncapping and Demannosylating Glycoproteins

Genetically engineered cells described herein can be used to producemolecular complexes having GAA activity. For example, geneticallyengineered cells can be used to produce molecule complexes having GAAactivity and comprising at least two polypeptides, each polypeptidehaving at least 85% sequence identity to a segment of the amino acidsequence set forth in SEQ ID NO: 1, each segment being derived byproteolysis of the amino acid sequence set forth in SEQ ID NO: 1 at oneor more sites between amino acid 50 and amino acid 74 (e.g., betweenamino acid 56 and amino acid 68 or between amino acid 60 and amino acid65). For example, a fungal cell can be engineered to include a nucleicacid encoding the amino acid sequence set forth in SEQ ID NO:1 and anucleic acid encoding an alkaline protease having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8 such thateach of the encoded polypeptides are secreted into the culture medium,where the alkaline protease can cleave the amino acid sequence set forthin SEQ ID NO:1. As described in Example 12, when the recombinant GAA wassecreted into the culture medium with the alkaline protease, processingof the 110 kDa precursor to the 95 kDa form was complete, i.e., the 110kDa precursor was not detected.

Genetically engineered cells described herein also can be used toproduce uncapped and demannosylated molecular complexes. Suchgenetically engineered cells can include a nucleic acid encoding apolypeptide having the amino acid sequence set forth in SEQ ID NO:1, anucleic acid encoding a mannosidase as described herein, and optionallya nucleic acid encoding an alkaline protease having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

A cell based method of producing uncapped and demannosylated moleculecomplexes can include introducing into a fungal cell geneticallyengineered to include a nucleic acid encoding a mannosidase that iscapable of hydrolyzing a mannose-1-phospho-6-mannose linkage or moietyto phospho-6-mannose, a nucleic acid encoding a polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 and optionally a nucleicacid encoding an alkaline protease having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, wherein the cellproduces the molecular complex described herein containing uncappedphosphorylated N-glycans. Such phosphorylated N-glycans can bedemannosylated as described above. In some embodiments, the nucleicacids encoding the mannosidase and GAA contain a secretion sequence suchthat the mannosidase and GAA are co-secreted. In genetically engineeredcells that include a nucleic acid encoding an alkaline protease, themolecular complexes can be processed to the 95 kDa form.

Another cell based method can include the steps of introducing into afungal cell genetically engineered to include a nucleic acid encoding amannosidase that is capable of (i) hydrolyzing amannose-1-phospho-6-mannose linkage or moiety to phospho-6-mannose and(ii) hydrolyzing a terminal alpha-1,2 mannose, alpha-1,3 mannose and/oralpha-1,6 mannose linkage or moiety of a phosphate containing glycan, anucleic acid encoding a polypeptide having the amino acid sequence setforth in SEQ ID NO: 1, and optionally a nucleic acid encoding analkaline protease having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO:8, wherein the cell producesuncapped and demannosylated molecular complexes. In some embodiments,the nucleic acids encoding the mannosidase and GAA contain a secretionsequence such that the mannosidase and target molecule are co-secreted.In genetically engineered cells that include a nucleic acid encoding analkaline protease, the molecular complexes can be processed to the 95kDa form.

Cells suitable for in vivo production of target molecules or molecularcomplexes can be of fungal origin, including Yarrowia lipolytica, Arxulaadeninivorans, methylotrophic yeast (such as a methylotrophic yeast ofthe genus Candida, Hansenula, Oogataea, Pichia or Torulopsis) orfilamentous fungi of the genus Aspergillus, Trichoderma, Neurospora,Fusarium, or Chrysosporium. Exemplary fungal species include, withoutlimitation, Pichia anomala, Pichia bovis, Pichia canadensis, Pichiacarsonii, Pichia farinose, Pichia fermentans, Pichia fluxuum, Pichiamembranaefaciens, Pichia membranaefaciens, Candida valida, Candidaalbicans, Candida ascalaphidarum, Candida amphixiae, Candida Antarctica,Candida atlantica, Candida atmosphaerica, Candida blattae, Candidacarpophila, Candida cerambycidarum, Candida chauliodes, Candidacorydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis,Candida fructus, Candida glabrata, Candida fermentati, Candidaguilliermondii, Candida haemulonii, Candida insectamens, Candidainsectorum, Candida intermedia, Candida jeffresii, Candida kefyr,Candida krusei, Candida lusitaniae, Candida lyxosophila, Candidamaltosa, Candida membranifaciens, Candida milleri, Candida oleophila,Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candidashehatea, Candida temnochilae, Candida tenuis, Candida tropicalis,Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candidaviswanathii, Candida utilis, Oogataea minuta, Pichia membranaefaciens,Pichia silvestris, Pichia membranaefaciens, Pichia chodati, Pichiamembranaefaciens, Pichia menbranaefaciens, Pichia minuscule, Pichiapastoris, Pichia pseudopolymorpha, Pichia quercuum, Pichia robertsii,Pichia saitoi, Pichia silvestrisi, Pichia strasburgensis, Pichiaterricola, Pichia vanriji, Pseudozyma Antarctica, Rhodosporidiumtoruloides, Rhodotorula glutinis, Saccharomyces bayanus, Saccharomycesbayanus, Saccharomyces momdshuricus, Saccharomyces uvarum, Saccharomycesbayanus, Saccharomyces cerevisiae, Saccharomyces bisporus, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces exiguous,Saccharomyces fermentati, Saccharomyces fragilis, Saccharomycesmarxianus, Saccharomyces mellis, Saccharomyces rosei, Saccharomycesrouxii, Saccharomyces uvarum, Saccharomyces willianus, Saccharomycodesludwigii, Saccharomycopsis capsularis, Saccharomycopsis fibuligera,Saccharomycopsis fibuligera, Endomyces hordei, Endomycopsis fobuligera.Saturnispora saitoi, Schizosaccharomyces octosporus, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporadelbrueckii, Saccharomyces dairensis, Torulaspora delbrueckii,Torulaspora fermentati, Saccharomyces fermentati, Torulasporadelbrueckii, Torulaspora rosei, Saccharomyces rosei, Torulasporadelbrueckii, Saccharomyces rosei, Torulaspora delbrueckii, Saccharomycesdelbrueckii, Torulaspora delbrueckii, Saccharomyces delbrueckii,Zygosaccharomyces mongolicus, Dorulaspora globosa, Debaryomycesglobosus, Torulopsis globosa, Trichosporon cutaneum, Trigonopsisvariabilis, Williopsis californica, Williopsis saturnus,Zygosaccharomyces bisporus, Zygosaccharomyces bisporus, Debaryomycesdisporua. Saccharomyces bisporas, Zygosaccharomyces bisporus,Saccharomyces bisporus, Zygosaccharomyces mellis, Zygosaccharomycespriorianus, Zygosaccharomyces rouxiim, Zygosaccharomyces rouxii,Zygosaccharomyces barkeri, Saccharomyces rouxii, Zygosaccharomycesrouxii, Zygosaccharomyces major, Saccharomyces rousii, Pichia anomala,Pichia bovis, Pichia Canadensis, Pichia carsonii, Pichia farinose,Pichia fermentans, Pichia fluxuum, Pichia membranaefaciens, Pichiapseudopolymorpha, Pichia quercuum, Pichia robertsii, PseudozymaAntarctica, Rhodosporidium toruloides, Rhodosporidium toruloides,Rhodotorula glutinis, Saccharomyces bayanus, Saccharomyces bayanus,Saccharomyces bisporus, Saccharomyces cerevisiae, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces fermentati,Saccharomyces fragilis, Saccharomycodes ludwigii, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporaglobosa, Trigonopsis variabilis, Williopsis californica, Williopsissaturnus, Zygosaccharomyces bisporus, Zygosaccharomyces mellis, orZygosaccharomyces rouxii. Exemplary filamentous fungi include variousspecies of Aspergillus including, but not limited to, Aspergilluscaesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillusclavatus, Aspergillus deflectus, Aspergillus flavus, Aspergillusfumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger,Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus,Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae,Aspergillus sydowi, Aspergillus tamari, Aspergillus terreus, Aspergillusustus, or Aspergillus versicolor. Such cells, prior to the geneticengineering as specified herein, can be obtained from a variety ofcommercial sources and research resource facilities, such as, forexample, the American Type Culture Collection (Rockville, Md.).

Genetic engineering of a cell can include, in addition to an exogenousnucleic acid encoding a mannosidase, GAA, and/or alkaline protease, oneor more genetic modifications such as: (i) deletion of an endogenousgene encoding an Outer CHain elongation (OCH1) protein; (ii)introduction of a recombinant nucleic acid encoding a polypeptidecapable of promoting mannosyl phosphorylation (e.g, a MNN4 polypeptidefrom Yarrowia lipolytica, S. cerevisiae, Ogataea minuta, Pichiapastoris, or C. albicans, or PNO1 polypeptide from P. pastoris) toincreasing phosphorylation of mannose residues; (iii) introduction orexpression of an RNA molecule that interferes with the functionalexpression of an OCH1 protein; (iv) introduction of a recombinantnucleic acid encoding a wild-type (e.g., endogenous or exogenous)protein having a N-glycosylation activity (i.e., expressing a proteinhaving an N-glycosylation activity); or (v) altering the promoter orenhancer elements of one or more endogenous genes encoding proteinshaving N-glycosylation activity to thus alter the expression of theirencoded proteins. RNA molecules include, e.g., small-interfering RNA(siRNA), short hairpin RNA (shRNA), anti-sense RNA, or micro RNA(miRNA). Genetic engineering also includes altering an endogenous geneencoding a protein having an N-glycosylation activity to produce aprotein having additions (e.g., a heterologous sequence), deletions, orsubstitutions (e.g., mutations such as point mutations; conservative ornon-conservative mutations). Mutations can be introduced specifically(e.g., by site-directed mutagenesis or homologous recombination) or canbe introduced randomly (for example, cells can be chemically mutagenizedas described in, e.g., Newman and Ferro-Novick (1987) J. Cell Biol.105(4):1587.

Genetic modifications described herein can result in one or more of (i)an increase in one or more activities in the genetically modified cell,(ii) a decrease in one or more activities in the genetically modifiedcell, or (iii) a change in the localization or intracellulardistribution of one or more activities in the genetically modified cell.It is understood that an increase in the amount of a particular activity(e.g., promoting mannosyl phosphorylation) can be due to overexpressingone or more proteins capable of promoting mannosyl phosphorylation, anincrease in copy number of an endogenous gene (e.g., gene duplication),or an alteration in the promoter or enhancer of an endogenous gene thatstimulates an increase in expression of the protein encoded by the gene.A decrease in one or more particular activities can be due tooverexpression of a mutant form (e.g., a dominant negative form),introduction or expression of one or more interfering RNA molecules thatreduce the expression of one or more proteins having a particularactivity, or deletion of one or more endogenous genes that encode aprotein having the particular activity.

To disrupt a gene by homologous recombination, a “gene replacement”vector can be constructed in such a way to include a selectable markergene. The selectable marker gene can be operably linked, at both 5′ and3′ end, to portions of the gene of sufficient length to mediatehomologous recombination. The selectable marker can be one of any numberof genes which either complement host cell auxotrophy or provideantibiotic resistance, including URA3, LEU2 and HIS3 genes. Othersuitable selectable markers include the CAT gene, which conferschloramphenicol resistance to yeast cells, or the lacZ gene, whichresults in blue colonies due to the expression of β-galactosidase.Linearized DNA fragments of the gene replacement vector are thenintroduced into the cells using methods well known in the art (seebelow). Integration of the linear fragments into the genome and thedisruption of the gene can be determined based on the selection markerand can be verified by, for example, Southern blot analysis. Aselectable marker can be removed from the genome of the host cell by,e.g., Cre-loxP systems (see below).

Alternatively, a gene replacement vector can be constructed in such away as to include a portion of the gene to be disrupted, which portionis devoid of any endogenous gene promoter sequence and encodes none oran inactive fragment of the coding sequence of the gene. An “inactivefragment” is a fragment of the gene that encodes a protein having, e.g.,less than about 10% (e.g., less than about 9%, less than about 8%, lessthan about 7%, less than about 6%, less than about 5%, less than about4%, less than about 3%, less than about 2%, less than about 1%, or 0%)of the activity of the protein produced from the full-length codingsequence of the gene. Such a portion of the gene is inserted in a vectorin such a way that no known promoter sequence is operably linked to thegene sequence, but that a stop codon and a transcription terminationsequence are operably linked to the portion of the gene sequence. Thisvector can be subsequently linearized in the portion of the genesequence and transformed into a cell. By way of single homologousrecombination, this linearized vector is then integrated in theendogenous counterpart of the gene.

Expression vectors can be autonomous or integrative. A recombinantnucleic acid (e.g., one encoding a mannosidase, GAA, or alkalineprotease) can be in introduced into the cell in the form of anexpression vector such as a plasmid, phage, transposon, cosmid or virusparticle. The recombinant nucleic acid can be maintainedextrachromosomally or it can be integrated into the yeast cellchromosomal DNA. Expression vectors can contain selection marker genesencoding proteins required for cell viability under selected conditions(e.g., URA3, which encodes an enzyme necessary for uracil biosynthesisor TRP1, which encodes an enzyme required for tryptophan biosynthesis)to permit detection and/or selection of those cells transformed with thedesired nucleic acids (see, e.g., U.S. Pat. No. 4,704,362). Expressionvectors can also include an autonomous replication sequence (ARS). Forexample, U.S. Pat. No. 4,837,148 describes autonomous replicationsequences which provide a suitable means for maintaining plasmids inPichia pastoris.

Integrative vectors are disclosed, e.g., in U.S. Pat. No. 4,882,279.Integrative vectors generally include a serially arranged sequence of atleast a first insertable DNA fragment, a selectable marker gene, and asecond insertable DNA fragment. The first and second insertable DNAfragments are each about 200 (e.g., about 250, about 300, about 350,about 400, about 450, about 500, or about 1000 or more) nucleotides inlength and have nucleotide sequences which are homologous to portions ofthe genomic DNA of the species to be transformed. A nucleotide sequencecontaining a gene of interest (e.g., a gene encoding GAA) for expressionis inserted in this vector between the first and second insertable DNAfragments whether before or after the marker gene. Integrative vectorscan be linearized prior to yeast transformation to facilitate theintegration of the nucleotide sequence of interest into the host cellgenome.

An expression vector can feature a recombinant nucleic acid under thecontrol of a yeast (e.g., Yarrowia lipolytica, Arxula adeninivorans, P.pastoris, or other suitable fungal species) promoter, which enables themto be expressed in fungal cells. Suitable yeast promoters include, e.g.,ADC1, TPI1, ADH2, hp4d, PDX, and Gal10 (see, e.g., Guarente et al.(1982) Proc. Natl. Acad. Sci. USA 79(23):7410) promoters. Additionalsuitable promoters are described in, e.g., Zhu and Zhang (1999)Bioinformatics 15(7-8):608-611 and U.S. Pat. No. 6,265,185.

A promoter can be constitutive or inducible (conditional). Aconstitutive promoter is understood to be a promoter whose expression isconstant under the standard culturing conditions. Inducible promotersare promoters that are responsive to one or more induction cues. Forexample, an inducible promoter can be chemically regulated (e.g., apromoter whose transcriptional activity is regulated by the presence orabsence of a chemical inducing agent such as an alcohol, tetracycline, asteroid, a metal, or other small molecule) or physically regulated(e.g., a promoter whose transcriptional activity is regulated by thepresence or absence of a physical inducer such as light or high or lowtemperatures). An inducible promoter can also be indirectly regulated byone or more transcription factors that are themselves directly regulatedby chemical or physical cues.

It is understood that other genetically engineered modifications canalso be conditional. For example, a gene can be conditionally deletedusing, e.g., a site-specific DNA recombinase such as the Cre-loxP system(see, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173 and U.S.Application Publication No. 20060014264).

A recombinant nucleic acid can be introduced into a cell describedherein using a variety of methods such as the spheroplast technique orthe whole-cell lithium chloride yeast transformation method. Othermethods useful for transformation of plasmids or linear nucleic acidvectors into cells are described in, for example, U.S. Pat. No.4,929,555; Hinnen et al. (1978) Proc. Nat. Acad. Sci. USA 75:1929; Itoet al. (1983) J. Bacteriol. 153:163; U.S. Pat. No. 4,879,231; andSreekrishna et al. (1987) Gene 59:115, the disclosures of each of whichare incorporated herein by reference in their entirety. Electroporationand PEG1000 whole cell transformation procedures may also be used, asdescribed by Cregg and Russel, Methods in Molecular Biology: PichiaProtocols, Chapter 3, Humana Press, Totowa, N.J., pp. 27-39 (1998).

Transformed fungal cells can be selected for by using appropriatetechniques including, but not limited to, culturing auxotrophic cellsafter transformation in the absence of the biochemical product required(due to the cell's auxotrophy), selection for and detection of a newphenotype, or culturing in the presence of an antibiotic which is toxicto the yeast in the absence of a resistance gene contained in thetransformants. Transformants can also be selected and/or verified byintegration of the expression cassette into the genome, which can beassessed by, e.g., Southern blot or PCR analysis.

Prior to introducing the vectors into a target cell of interest, thevectors can be grown (e.g., amplified) in bacterial cells such asEscherichia coli (E. coli) as described above. The vector DNA can beisolated from bacterial cells by any of the methods known in the artwhich result in the purification of vector DNA from the bacterialmilieu. The purified vector DNA can be extracted extensively withphenol, chloroform, and ether, to ensure that no E. coli proteins arepresent in the plasmid DNA preparation, since these proteins can betoxic to mammalian cells.

In some embodiments, the genetically engineered fungal cell lacks theOCH1 gene or gene products (e.g., mRNA or protein) thereof, and isdeficient in OCH1 activity. In some embodiments, the geneticallyengineered cell expresses a polypeptide capable of promoting mannosylphosphorylation (e.g., a MNN4 polypeptide from Yarrowia lipolytica, S.cerevisiae, Ogataea minuta, Pichia pastoris, or C. albicans, or a PNO1polypeptide from P. pastoris). For example, the fungal cell can expressa MNN4 polypeptide from Y. lipolytica (Genbank® Acccession Nos:XM_(—)503217, Genolevures Ref: YALI0D24101g). In some embodiments, thegenetically engineered cell is deficient in OCH1 activity and expressesa polypeptide capable of promoting mannosyl phosphorylation.

Following uncapping and demannosylation, the molecular complex can beisolated. In some embodiments, the molecular complex is maintainedwithin the yeast cell and released upon cell lysis. In some embodiments,the molecular complex is secreted into the culture medium via amechanism provided by a coding sequence (either native to the exogenousnucleic acid or engineered into the expression vector), which directssecretion of the molecule from the cell. The presence of the uncappedand demannosylated molecular complex in the cell lysate or culturemedium can be verified by a variety of standard protocols for detectingthe presence of the molecule, e.g., immunoblotting orradioimmunoprecipitation with an antibody specific for GAA, or testingfor a specific enzyme activity (e.g., glycogen degradation).

In some embodiments, following isolation, the uncapped anddemannosylated target molecule or molecular complex can be attached to aheterologous moiety, e.g., using enzymatic or chemical means. A“heterologous moiety” refers to any constituent that is joined (e.g.,covalently or non-covalently) to the altered target molecule, whichconstituent is different from a constituent originally present on thealtered target molecule. Heterologous moieties include, e.g., polymers,carriers, adjuvants, immunotoxins, or detectable (e.g., fluorescent,luminescent, or radioactive) moieties. In some embodiments, anadditional N-glycan can be added to the altered target molecule.

Methods for detecting glycosylation of molecules include DNAsequencer-assisted (DSA), fluorophore-assisted carbohydrateelectrophoresis (FACE) or surface-enhanced laser desorption/ionizationtime-of-flight mass spectrometry (SELDI-TOF MS). For example, ananalysis can utilize DSA-FACE in which, for example, glycoproteins aredenatured followed by immobilization on, e.g., a membrane. Theglycoproteins can then be reduced with a suitable reducing agent such asdithiothreitol (DTT) or β-mercaptoethanol. The sulfhydryl groups of theproteins can be carboxylated using an acid such as iodoacetic acid.Next, the N-glycans can be released from the protein using an enzymesuch as N-glycosidase F. N-glycans, optionally, can be reconstituted andderivatized by reductive amination. For example, the released N-glycanscan be labeled with a fluorophore such APTS(8-aminopyrene-1,3,6-trisulfonic acid), at the reducing terminus byreductive amination. The stoichiometry of labeling is such that only oneAPTS molecule is attached to each molecule of oligosaccharide. Thederivatized N-glycans can then be concentrated. Instrumentation suitablefor N-glycan analysis includes, e.g., the ABI PRISM® 377 DNA sequencer(Applied Biosystems). Data analysis can be performed using, e.g.,GENESCAN® 3.1 software (Applied Biosystems). Isolated mannoproteins canbe further treated with one or more enzymes such as calf intestinephosphatase to confirm their N-glycan status. Additional methods ofN-glycan analysis include, e.g., mass spectrometry (e.g., MALDI-TOF-MS),high-pressure liquid chromatography (HPLC) on normal phase, reversedphase and ion exchange chromatography (e.g., with pulsed amperometricdetection when glycans are not labeled and with UV absorbance orfluorescence if glycans are appropriately labeled). See also Callewaertet al. (2001) Glycobiology 11(4):275-281 and Freire et al. (2006)Bioconjug. Chem. 17(2):559-564.

Cultures of Engineered Cells

This document also provides a substantially pure culture of any of thegenetically engineered cells described herein. As used herein, a“substantially pure culture” of a genetically engineered cell is aculture of that cell in which less than about 40% (i.e., less thanabout: 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%;0.01%; 0.001%; 0.0001%; or even less) of the total number of viablecells in the culture are viable cells other than the geneticallyengineered cell, e.g., bacterial, fungal (including yeast), mycoplasmal,or protozoan cells. The term “about” in this context means that therelevant percentage can be 15% percent of the specified percentage aboveor below the specified percentage. Thus, for example, about 20% can be17% to 23%. Such a culture of genetically engineered cells includes thecells and a growth, storage, or transport medium. Media can be liquid,semi-solid (e.g., gelatinous media), or frozen. The culture includes thecells growing in the liquid or in/on the semi-solid medium or beingstored or transported in a storage or transport medium, including afrozen storage or transport medium. The cultures are in a culture vesselor storage vessel or substrate (e.g., a culture dish, flask, or tube ora storage vial or tube).

The genetically engineered cells described herein can be stored, forexample, as frozen cell suspensions, e.g., in buffer containing acryoprotectant such as glycerol or sucrose, as lyophilized cells.Alternatively, they can be stored, for example, as dried cellpreparations obtained, e.g., by fluidized bed drying or spray drying, orany other suitable drying method.

Pharmaceutical Compositions and Methods of Treatment

GAA molecules and molecular complexes described herein, e.g., molecularcomplexes containing at least one modification that enhances transportto the interior of a mammalian cell, can be incorporated into apharmaceutical composition containing a therapeutically effective amountof the molecule and one or more adjuvants, excipients, carriers, and/ordiluents. Acceptable diluents, carriers and excipients typically do notadversely affect a recipient's homeostasis (e.g., electrolyte balance).Acceptable carriers include biocompatible, inert or bioabsorbable salts,buffering agents, oligo- or polysaccharides, polymers,viscosity-improving agents, preservatives and the like. One exemplarycarrier is physiologic saline (0.15 M NaCl, pH 7.0 to 7.4). Anotherexemplary carrier is 50 mM sodium phosphate, 100 mM sodium chloride.Further details on techniques for formulation and administration ofpharmaceutical compositions can be found in, e.g., Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).Supplementary active compounds can also be incorporated into thecompositions.

Administration of a pharmaceutical composition containing molecularcomplexes with one or modifications described herein can be systemic orlocal. Pharmaceutical compositions can be formulated such that they aresuitable for parenteral and/or non-parenteral administration. Specificadministration modalities include subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, intrathecal, oral, rectal,buccal, topical, nasal, ophthalmic, intra-articular, intra-arterial,sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterineadministration.

Administration can be by periodic injections of a bolus of thepharmaceutical composition or can be uninterrupted or continuous byintravenous or intraperitoneal administration from a reservoir which isexternal (e.g., an IV bag) or internal (e.g., a bioerodable implant, abioartificial organ, or a colony of implanted altered N-glycosylationmolecule production cells). See, e.g., U.S. Pat. Nos. 4,407,957,5,798,113, and 5,800,828. Administration of a pharmaceutical compositioncan be achieved using suitable delivery means such as: a pump (see,e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer, 41:1270 (1993);Cancer Research, 44:1698 (1984); microencapsulation (see, e.g., U.S.Pat. Nos. 4,352,883; 4,353,888; and 5,084,350); continuous releasepolymer implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666);macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsWO92/19195, WO 95/05452); injection, either subcutaneously,intravenously, intra-arterially, intramuscularly, or to other suitablesite; or oral administration, in capsule, liquid, tablet, pill, orprolonged release formulation.

Examples of parenteral delivery systems include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, pumpdelivery, encapsulated cell delivery, liposomal delivery,needle-delivered injection, needle-less injection, nebulizer,aerosolizer, electroporation, and transdermal patch.

Formulations suitable for parenteral administration conveniently containa sterile aqueous preparation of the altered N-glycosylation molecule,which preferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Formulations can be presented inunit-dose or multi-dose form.

Formulations suitable for oral administration can be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the altered N-glycosylationmolecule; or a suspension in an aqueous liquor or a non-aqueous liquid,such as a syrup, an elixir, an emulsion, or a draught.

A molecular complex containing at least one modification that enhancestransport of the complex to the interior of a mammalian cell that issuitable for topical administration can be administered to a mammal(e.g., a human patient) as, e.g., a cream, a spray, a foam, a gel, anointment, a salve, or a dry rub. A dry rub can be rehydrated at the siteof administration. Such molecules can also be infused directly into(e.g., soaked into and dried) a bandage, gauze, or patch, which can thenbe applied topically. Such molecules can also be maintained in asemi-liquid, gelled, or fully-liquid state in a bandage, gauze, or patchfor topical administration (see, e.g., U.S. Pat. No. 4,307,717).

Therapeutically effective amounts of a pharmaceutical composition can beadministered to a subject in need thereof in a dosage regimenascertainable by one of skill in the art. For example, a composition canbe administered to the subject, e.g., systemically at a dosage from 0.01μg/kg to 10,000 μg/kg body weight of the subject, per dose. In anotherexample, the dosage is from 1 μg/kg to 100 μg/kg body weight of thesubject, per dose. In another example, the dosage is from 1 μg/kg to 30μg/kg body weight of the subject, per dose, e.g., from 3 μg/kg to 10μg/kg body weight of the subject, per dose.

In order to optimize therapeutic efficacy, a molecular complex describedherein can be first administered at different dosing regimens. The unitdose and regimen depend on factors that include, e.g., the species ofmammal, its immune status, the body weight of the mammal. Typically,levels of such a molecular complex in a tissue can be monitored usingappropriate screening assays as part of a clinical testing procedure,e.g., to determine the efficacy of a given treatment regimen.

The frequency of dosing for a molecular complex described herein iswithin the skills and clinical judgement of medical practitioners (e.g.,doctors or nurses). Typically, the administration regime is establishedby clinical trials which may establish optimal administrationparameters. However, the practitioner may vary such administrationregimes according to the subject's age, health, weight, sex and medicalstatus. The frequency of dosing can be varied depending on whether thetreatment is prophylactic or therapeutic.

Toxicity and therapeutic efficacy of such molecular complexes orpharmaceutical compositions thereof can be determined by knownpharmaceutical procedures in, for example, cell cultures or experimentalanimals. These procedures can be used, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Pharmaceutical compositionsthat exhibit high therapeutic indices are preferred. Whilepharmaceutical compositions that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to normal cells (e.g., non-target cells) and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in appropriate subjects(e.g., human patients). The dosage of such pharmaceutical compositionslies generally within a range of circulating concentrations that includethe ED₅₀ with little or no toxicity. The dosage may vary within thisrange depending upon the dosage form employed and the route ofadministration utilized. For a pharmaceutical composition used asdescribed herein (e.g., for treating a metabolic disorder in a subject),the therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the pharmaceutical composition which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

As defined herein, a “therapeutically effective amount” of a molecularcomplex is an amount of the complex that is capable of producing amedically desirable result (e.g., amelioration of one or more symptomsof Pompe's disease) in a treated subject. A therapeutically effectiveamount (i.e., an effective dosage) can includes milligram or microgramamounts of the complex per kilogram of subject or sample weight (e.g.,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram).

The subject can be any mammal, e.g., a human (e.g., a human patient) ora non-human primate (e.g., chimpanzee, baboon, or monkey), a mouse, arat, a rabbit, a guinea pig, a gerbil, a hamster, a horse, a type oflivestock (e.g., cow, pig, sheep, or goat), a dog, a cat, or a whale.

A molecular complex or pharmaceutical composition thereof describedherein can be administered to a subject as a combination therapy withanother treatment used for Pompe's disease. For example, the combinationtherapy can include administering to the subject (e.g., a human patient)one or more additional agents that provide a therapeutic benefit to thesubject who has, or is at risk of developing (e.g., due to a mutation inthe gene encoding GAA) Pompe's disease. Thus, the compound orpharmaceutical composition and the one or more additional agents can beadministered at the same time. Alternatively, the molecular complex canbe administered first and the one or more additional agents administeredsecond, or vice versa.

Any of the molecular complexes described herein can be lyophilized.

Any of the pharmaceutical compositions described herein can be includedin a container, pack, or dispenser together with instructions foradministration. In some embodiments, the composition is packaged as asingle use vial.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Uncapping and De-Mannosylation of Recombinant huGAAwith CcMan5 and Jack Bean α-Mannosidase

Recombinant human GAA (rhGAA) was produced as described in WO2011/039634using Y. lipolytica production strain OXYY1589, which contains threecopies of the human alpha glucosidase gene (also known as acid alphaglucosidase or acid maltase EC3.2.1.3) and two copies of the Y.lipolytica MNN4 gene. The amino acid sequence of human GAA is set forthin FIG. 1. The genotype of strain OXY1589 is as follows:

MatA, leu2-958, ura3-302, xpr2-322,gut2-744, ade2-844POX2-Lip2pre-huGAA:URA3Ex::zetaPOX2-Lip2pre-huGAA:LEU2Ex::zetaPOX2-Lip2pre-hGM-CSF: GUTEx::zetaY1MNN4-POX2-hp4d-YLMNN4:ADE2::PT targeted

RhGAA was uncapped and demannosylated with Cellulosimicrobium cellulansmannosidase (CcMan5) and Jack bean a mannosidase (JbMan) (Sigma ProductM7257, 3.0 M ammonium sulphate suspension). CcMan5 was producedrecombinantly by first cloning the nucleic acid encoding the CcMan5polypeptide (FIG. 2A) into vector pLSAH36, which contains a DsbA signalsequence and results in the expression of a protein with an N-terminalHIS tag. FIGS. 2B and 2C contain the amino acid sequence of the CcMan5polypeptide with and without signal sequence, respectively. PlasmidpLSAH36 was cloned into E. coli B21 cells and proteins residing in theperiplasm were isolated and purified using a Talon column. Before usingthe ammonium sulphate suspension of JbMan, it was further purified bygel filtration through a Superdex 200 column to remove contaminatingphosphatase activities.

RhGAA (concentration of about 5 mg/mL in 20 mM sodium acetate (NaOAc)buffer, pH 5.0) was uncapped and demannosylated by incubating withCcMan5 (about 0.15-0.30 mg/mL in phosphate buffered saline (PBS)) andJbMan (about 0.5-1 mg/mL in PBS) in a w:w ratio of 100:5:10 forhuGAA:CcMan5:JbMan. The total reaction volume was diluted with 500 mMNaOAc buffer, pH 5.0 and 100 mM CaCl₂ to obtain final concentrations of100 mM NaOAc and 2 mM CaCl₂. The reaction mixture was incubated at 30°C. for 16 hours.

To evaluate the uncapping process and to analyze the N-glycan profile ofthe purified huGAA, the N-glycans of 5 μg glycoprotein were releasedwith N-Glycosidase F (PNGaseF), labeled with APTS(8-amino-1,3,6-pyrenetrisulfonic acid; trisodium salt) and subsequentlyanalyzed on DSA-FACE (DNA Sequencer-Aided Fluorophore-AssistedCarbohydrate Electrophoresis). The method essentially follows theprotocol described in Laroy et al, Nature Protocols, 1:397-405 (2006).

The DSA-FACE electropherograms of the N-glycans from huGAA (76 kD form)before (panel B) and after (panel C) treatment with CcMan5 and JbMan arepresented in FIG. 3A. Panel A is a reference sample containing theN-glycans released from RNaseB with PNGaseF. The N-glycan mixturereleased from capped huGAA is mainly composed of ManP-Man₈GlcNAc₂ and(ManP)₂-Man₈GlcNAc₂ (FIG. 3A, panel B). A peak running slightly fasterthan ManP-Man₈GlcNAc₂ can be assigned to ManP-Man₇GlcNAc₂. The mainpeaks observed after uncapping and demannosylation can be assigned tothe double phosphorylated P₂-Man₆GlcNAc₂ and the monophosphorylatedP-Man₄GlcNAc₂, P-Man₅GlcNAc₂ and P-Man₆GlcNAc₂ (Panel C).

The uncapping of different processed forms of huGAA results in the sameN-glycan profiles (FIG. 3B) for the 76 kD form (Panel B), 95 kD form(Panel C) and 110 kD form (Panel D).

Example 2 Purification of 110 kDa rhGAA

The 110 kDa form of rhGAA was isolated from strain OXYY1589 as follows.After harvest, the broth was centrifuged and filtered using a Duraporemembrane (Merck Millipore). Ammonium sulphate (AMS) was added to aconcentration of 1 M and the solute was filtered before loading on ahydrophobic interaction chromatography (HIC) column, equilibrated in 20mM sodium phosphate pH 6, 1 M ammonium sulphate. The product was elutedwith 20 mM sodium phosphate pH 6.

Before loading on a second chromatography column, the product was firstconcentrated via tangential flow filtration (TFF) on a regeneratedcellulose membrane, then exchanged from buffer to 20 mM sodium acetatepH 4.5. This material was loaded on a cation exchange chromatography(CEX) column, equilibrated with 20 mM sodium acetate pH 4.5. Afterloading the column, it was washed with equilibration buffer until the UVabsorbance signal reached baseline, and then washed with 20 mM sodiumacetate pH 4.5, 50 mM NaCl. The product was eluted in 20 mM sodiumacetate pH 4.5, 150 mM NaCl, and concentrated and exchanged from bufferto 20 mM sodium acetate pH 5. (See below)

The sample was uncapped and demannosylated as described in Example 1then D-mannitol was added to a concentration of 100 mM. Three quartersof that material was reduced in volume via TFF using a regeneratedcellulose membrane having a 10 kDa molecular weight cut off (MWCO) andpurified further via size exclusion chromatography (SEC) on a Superdex200 column equilibrated at 4° C. with 25 mM sodium phosphate pH 6, 150mM NaCl, 100 mM D-mannitol. Fractions were screened afterwards forpurity on cibacron-blue stained polyacrylamide gels under denaturatingconditions. Pooled fractions were concentrated via TFF andultracentrifuged using Amicon centrifugal filters of 10 kD MWCO(regenerated cellulose membrane, Merck Millipore).

Example 3 Purification of 110 kDa rhGAA

The 110 kDa form of rhGAA was isolated from strain OXYY1589 as follows.After harvest, the material was centrifuged and filtered before theconcentration of AMS was increased to 1 M. The solute was again filteredand the product was captured on a HIC column, equilibrated with 20 mMsodium phosphate pH 6, 1 M AMS, and released in a step gradient from 1to 0 M AMS in a 20 mM sodium phosphate pH 6 buffer.

The eluate was concentrated and buffer exchanged to 10 mM BIS-TRIS, pH 6via TFF on a Vivaflow 200 module (PES membrane, 10 kD MWCO, Sartorius).The desalted material was brought onto an anion exchange chromatography(AEC) column. After washing of the column until the UV signal almostreached baseline, a two-phase continuous salt gradient was applied; thefirst gradient going from 0 to 0.3 M NaCl, the second from 0.3 to 1 MNaCl. Fractions were collected during the gradient and screened for GAAvia a qualitative 4-methylumbellifferyl-α-D-glucopyranoside (4-MUG). Inthe 4-MUG assay, reactions were started by adding a reaction bufferconsisting of 0.35 mM 4-MUG, 0.1% BSA and 100 mM sodium acetate pH 4 ina 10:1 volume proportion to 10 μl of elution fraction. After incubatingfor 30 minutes to 1 hour at 37° C., an equal volume of 100 mM glycine pH11 was added to stop the reaction. The release of the fluorogenicreaction product 4-methylumbelliferone was observed under UV-light.

Fractions containing GAA were pooled and concentrated via TFF on aVivaflow 200 module (PES membrane, 10 kD MWCO, Sartorius) andultracentrifugation using Amicon centrifugal filters of 10 kD MWCO(regenerated cellulose membrane, Merck Millipore).

The concentrated material was split in two and purified further on aSuperdex 200 column equilibrated at 4° C. with 50 mM sodium phosphate pH6, 250 mM NaCl, 0.05% Tween-20. Fractions were screened afterwards forpurity on cibacron-blue stained polyacrylamide gels under denaturingconditions, and phosphatase content was determined using a colormetrictest using para-nitrophenylphosphate, which measures the enzymaticrelease of the yellow colored p-nitrophenolate reaction product at awavelength of 405 nm.

Pilot pools were made from fractions containing GAA. The total proteinof the pilot pools was determined via the Bradford assay. Selectedfractions were pooled for concentration onto a Vivaflow 200 TFF module(PES membrane, 10 kD MWCO, Sartorius). The volume was further reducedusing 15 ml Amicon centrifugal filters of 10 kD MWCO (regeneratedcellulose membrane, Merck Millipore).

The concentrated material was subjected to a second round of sizeexclusion chromatography (SEC) using the same conditions as for thefirst SEC step. Fractions were again screened for purity oncibacron-blue stained polyacrylamide gels under denaturing conditions.Fractions were pooled according to the chosen pilot pool andconcentrated on 15 ml Amicon centrifugal filters (10 kD MWCO,regenerated cellulose membrane, Merck Millipore).

Example 4 Purification of 95 kDa rhGAA

The 95 kDa form of rhGAA was isolated from strain OXYY1589 as follows.

After harvest, the broth was centrifuged and filtered using a Duraporemembrane (Merck Millipore). The product was afterwards concentrated viaTFF on a modified polyethersulfone (PES) membrane with amolecular-weight-cut-off (MWCO) of 10 kD. AMS was added to aconcentration of 1 M and the solute was filtered before loading on a HICcolumn, equilibrated in 20 mM sodium phosphate pH 6, 1 M AMS. Theproduct was eluted with water, the pH of the eluate was adjusted byadding a stock buffer of 100 mM BIS-TRIS pH 6 to a concentration of 10mM, and it was stored at 4° C. for 13 days.

Before loading on an AEX column, the product was concentrated via TFF ona regenerated cellulose membrane with an MWCO of 10 kD andbuffer-exchanged to 10 mM BIS-TRIS pH 6. The desalted material wasprocessed further via AEX chromatography, performed as described inExample 3. Fractions were collected during the gradient and screened forGAA via the qualitative 4-MUG assay. Fractions containing GAA werepooled for further purification.

For the third chromatography step, the concentration of AMS wasincreased to 1 M, and, after filtration, the material was furtherpurified via HIC. A continuous salt gradient from 1 to 0 M AMS wasapplied while collecting fractions during the gradient. All fractionswere screened for GAA via the qualitative assay and those containing GAAwere pooled for further processing.

The pool was concentrated via ultra-centrifugation using 15 ml Amiconcentrifugal filters of 10 kD MWCO regenerated cellulose membrane andfurther purified via SEC using the same procedures as described inExample 3. Fractions were screened afterwards for purity oncibacron-blue stained polyacrylamide gels under denaturing conditions.The 90% pure GAA fractions were pooled and first concentrated on a TFFVivaflow 200 module (PES membrane, 10 kD MWCO, Sartorius), and thensubjected to ultra-centrifugation using 15 ml Amicon centrifugal filters(10 kD MWCO, regenerated cellulose membrane, Merck Millipore). Theconcentrated material was subjected to a second round of SEC using thesame conditions as for the first SEC step. Fractions were screened forGAA using the qualitative 4-MUG GAA assay. Fractions having GAA activitywere pooled and concentrated.

After uncapping and demannosylation as set forth in Example 1,D-mannitol was added to a concentration of 100 mM and the volume wasagain reduced before loading onto a final Superdex 200 gel filtrationcolumn, equilibrated at 4° C. with 25 mM sodium phosphate pH 6, 150 mMNaCl, 100 mM D-mannitol. Fractions were screened for GAA using the 4-MUGqualitative assay, and those containing the product were pooled andconcentrated.

Example 5 Purification of 95-110 kDa rhGAA Mix

Both the 110 kDa precursor and 95 kDa form of rhGAA was isolated fromstrain OXYY1589 as follows. After harvest, the material was processed tothe second chromatography step as described in Example 2. After the HICstep, the product was concentrated and the buffer exchanged to 10 mMBIS-TRIS pH 6 via TFF, and loaded on an AEX column. The product waseluted in a single step from 0 to 300 mM NaCl at pH 6 (10 mM BIS-TRIS)and then concentrated using a Pellicon XL50 TFF module (regeneratedcellulose membrane with a 10 kD MWCO). Half of the material was furtherpurified via size exclusion chromatography. The chromatography step wasperformed as described in Example 3, but the selection of the fractionsfor further processing was only done on the basis of purity oncibacron-blue stained polyacrylamide gels under denaturing conditions.

Half of the pool was concentrated and combined with the remainder of theAEX-material. After uncapping and demannosylation, the concentration ofD-mannitol was increased to 100 mM and the subsequent concentration andSEC steps were done following the same procedures as described inExample 2. Fractions were pooled on the basis of the 4-MUG qualitativeassay and pooled with uncapped product from Example 6.

Example 6 Purification of 95 kDa rhGAA

The 95 kDa form of rhGAA was isolated from strain OXYY1589 as follows.After harvest, the material was processed up to and including the AEXstep as described in Example 3. In the AEX step, a significant amount ofthe product resided in the flow through fraction due to an increase ofconductivity during the loading. The flow through material was thereforeagain diafiltered to the appropriate buffer and subjected to a secondround of AEX chromatography. Both amounts (batch A and batch B) werefrom here on processed separately.

Batch A was combined with the remainder of the SEC pool from Example 5and the remainder of the CEX pool from Example 2 and the poolsubsequently concentrated and diafiltered to a buffer containing 10 mMBIS-TRIS pH 6, 300 mM NaCl. The material was incubated at 30° C. for 65h. The pH then was lowered to pH 5 by adding a 1 M stock buffer ofsodium acetate pH 5 to a concentration of 125 mM, and the sample wasagain incubated at 30° C. After 24 h, the product was treated withFlavourzyme (Novozymes Corp), a protease mix from Aspergillus oryzae,using a 40:1 weight:weight ratio total protein content of the productversus protease mix, and was for the last time incubated at 30° C. Afteran overnight incubation, the material was purified via a first SEC step,performed under the same conditions as described in Example 3. Fractionswere pooled that were estimated to contain pure product on the basis ofcibacron-blue stained polyacrylamide gels under denaturing conditions.After concentration and buffer exchange to 20 mM sodium acetate pH 5,the material was uncapped and demannosylated as set forth in Example 1.D-mannitol was added to a concentration of 100 mM and the material waspooled with uncapped and demannosylated material from Example 5. Thefinal SEC step and subsequent sample manipulations were performed asdescribed in Example 2.

Batch B was pooled with end material from Example 3 and the pool wasconcentrated and diafiltered to a buffer containing 10 mM BIS-TRIS pH 6,300 mM NaCl. The product was then treated with the A. oryzae proteasemix for an overnight incubation period at 30° C. using the same weightratios as described in Example 5, and, afterwards, purified via a firstSEC step, performed under the same conditions as described in Example 3.Further processing of the product was done as described in Example 5.

In the final batch, product from batch A and batch B were mixed in 14:1ratio in GAA content.

Example 7 Purification of 76 kDa rhGAA

The 76 kDa form of rhGAA was isolated from strain OXYY1589 as follows.After harvest, the culture was subjected to two rounds of continuouscentrifugation. The supernatant was pooled and AMS was introduced to aconcentration of approximately 1 M. After dissolution, 1 volume of HICresin, pre-equilibrated in 20 mM sodium phosphate pH 6, 1 M AMS, wasadded to 50 volumes of supernatant while stirring to bind the product ina batch uptake mode. The resulting slurry was stored overnight at 4° C.without stirring. During this period, a brown colored layer settled atthe top of the solute that was removed in the morning via gentleaspiration. The resin was washed three times with three volumes of leadbuffer (20 mM sodium phosphate pH 6, 1 M AMS) in each round before itwas packed into a column. The packed resin was washed until UV signalalmost reached baseline and the product was afterwards eluted withwater. The pH of the eluate was adjusted by adding a stock buffer of 100mM BIS-TRIS pH 6 to a concentration of 10 mM. The material was thensterile filtered in a bag and stored for a period of eleven days at 4°C.

The second and third chromatography steps and accompanying manipulationsof the material were performed as described in Example 4. The pool afterthe third chromatography step was first concentrated approximately seventimes on two TFF Vivaflow 200 modules coupled in parallel (PES membrane,10 kD MWCO, Sartorius), and then ultra-centrifuged using 15 ml Amiconcentrifugal filters of 10 kD MWCO (regenerated cellulose membrane, MerckMillipore). The concentrated material was split in two and purifiedfurther via SEC using the same conditions as described for Example 4.Fractions were screened afterwards for purity on cibacron-blue stainedpolyacrylamide gels under denaturing conditions. Selected fractions werepooled for concentration onto two Vivaflow 200 TFF modules coupled inparallel (PES membrane, 10 kD MWCO, Sartorius). The volume was furtherreduced using 15 ml Amicon centrifugal filters of 10 kD MWCO(regenerated cellulose membrane, Merck Millipore).

After uncapping and demannosylation, D-mannitol was added to aconcentration of 100 mM and the sample was again concentrated on aVivaflow 50 TFF module (PES membrane, 10 kD MWCO, Sartorius) beforeloading onto a final SEC column, performed in the same way as describedin Example 4. Product containing fractions were pooled and concentrated.

Example 8 Enzymatic Characterization of the Different Variants of huGAA(76, 95 and 110 kD Variants) Using the Artificial Chromogenic Substratep-Nitrophenyl-α-D-Glucopyranoside

The artificial chromogenic substrate p-nitrophenyl-α-D-glucopyranoside(PNPG) was used to determine the kinetic parameters of the unprocessedhuGAA (110 kDa) obtained in Example 2 and the processed huGAA variantsobtained in Example 7 (76 kDa), Example 6 (95 kDa) and Example 4 (95kDa) A comparison also was made with the commercial human α-glucosidase,Myozyme® (alglucosidase alpha, Genzyme).

The enzymes were diluted to 20 μg/ml in 100 mM sodium acetate buffer pH4.0, containing 0.1% BSA and 100 mM AMS (reaction buffer). Ten μl of theenzyme solutions were added to a 96-well plate in triplicate. The PNPGsubstrate (Sigma) was diluted to various substrate concentrations (10,6, 4, 2, 1.6, 1.2, 0.8, and 0.4 mM) in reaction buffer and 90 μl of thediluted substrate was added to each well. The enzymatic reaction wasincubated for 60 min at 37° C. followed by the addition of 100 μl 10%sodium carbonate, pH 12 to quench the reaction. The absorbance wasmeasured at 405 nm. A standard curve with p-nitrophenol (PNP) wasmeasured to calculate the amount of product formed per minute. Thevelocity expressed as μM/min was plotted in function of the differentsubstrate concentrations generating a Michaelis-Menten curve. GraphPadPrism was used to calculate the Vmax and Km according to a direct fit tothe Michaelis-Menten equation. The catalytic constant kcat and thecatalytic efficiency kcat/Km were calculated. The specific activity ofthe various enzymes was reported as U/mg where 1 unit is defined as theamount of enzyme that catalyzes the hydrolysis of 1 nmol substrate perminute at 2 mM substrate concentration in 100 mM sodium acetate buffer,pH 4.0+0.1% BSA and 100 mM AMS. The results are shown in Table 1.

TABLE 1 95 kDa 76 kDa 110 kDa 95 kDa Myozyme (Ex. 4) (Ex. 7) (Ex. 2)(Ex. 6) Vmax 12 12 14 13 13 (μM/min) Km (mM) 4.4 4.4 4.3 4.4 4.7 kcat(min⁻¹) 660 677 770 688 730 kcat/Km 150 154 179 156 155 (min⁻¹mM−¹) Sp.Activity 2000 1910 2415 1935 1980 (U/mg)

The unprocessed and processed forms of huGAA and Myozyme have comparablekinetic parameters towards the substrate PNPG. This is in accordancewith data reported in literature for human acid α-glucosidase from Mousemilk and Chinese hamster ovary (CHO) medium (Bijvoet et at (1998), HumanMolecular Genetics, 7, 1815-1824). The unprocessed (110 kD) and theprocessed (76 kD) form were reported to have the same Km and kcat valuefor the artificial substrate 4-methylumbelliferyl-α-D-glucopyranoside.

Example 9 Enzymatic Characterization of the Different Variants of huGAA(76, 95 and 110 kD Variants) Using Rabbit Liver Glycogen as theSubstrate

The enzymatic parameters of the unprocessed huGAA (110 kD variant;Example 2) and the processed huGAA variants (76 kDa, Example 7; and 95kD, Example 6) were tested using rabbit liver glycogen (lot N°099K37931V, Sigma). A comparison was made with the commercial humanα-glucosidase, Myozyme® (alglucosidase alpha, Genzyme). The enzymes werediluted to 500 ng/ml in 100 mM sodium acetate buffer pH 4.0. 50 μl ofthe enzyme solutions were added to a 96-well plate in duplicate. Theglycogen substrate was diluted to various substrate concentrations (250,200, 150, 100, 75, 50, 25 mg/ml) in acetate buffer and 100 μl of thediluted substrate was added to each well. The enzymatic reaction wasincubated for 60 min at 37° C. The amount of glucose was measured usingthe glucose-oxidase method with the amplex red substrate.

A glucose standard curve was measured to calculate the amount of productformed per minute. The enzyme velocity expressed as μM/min was plottedin function of the different substrate concentrations generating aMichaelis-Menten curve. GraphPad Prism was used to calculate the Vmax,and Km according to a direct fit to the Michaelis-Menten equation. Thecatalytic constant kcat and the catalytic efficiency kcat/Km werecalculated. The specific activity of the various enzymes was reported asU/mg where 1 unit is defined as the amount of enzyme that catalyses theformation of 1 μmol glucose per minute at 50 mg/ml final substrateconcentration in 100 mM sodium acetate buffer, pH 4.0. The results areshown in Table 2.

TABLE 2 76 kDa 95 kDa 110 kDa Myozyme (Ex. 7) (Ex. 6) (Ex. 2) Vmax 32 ±6  15 ± 2 13 ± 1 11 ± 1 (μM/min) Km (mM) 600 ± 140 100 ± 10 93 ± 8 162 ±17 kcat (min⁻¹) 21100 10000 8600 7260 kcat/Km 35 100 92 45 (min⁻¹mM−¹)Sp. Activity 14 32 27 16 (U/mg)

In this experiment substrate saturation cannot be reached due to thelimited solubility of rabbit glycogen (FIG. 4). For Myozyme, only anapparent Km and kcat value were calculated. For the three huGAAvariants, lower apparent Km values were determined. The catalyticefficiency of the processed forms is two fold higher than the catalyticefficiency of unprocessed huGAA and Myozyme.

Example 10 Effect of Acid Alpha Glucosidase on Glycogen Clearance in aMouse Model of Pompe Disease

The GAA products from Example 7 (76 kDa, uncapped and demannosylated),Example 6 (95 kDa, uncapped and demannosylated), and Example 2 (110 kDa,uncapped and demannosylated) were administered to a mouse model ofPompe's disease to determine the glycogen clearance from skeletal muscleand heart.

The experiment was performed with FVB GAA knockout mice and FVB wildtype mice. This animal model was chosen as a test system since it is agood representative for the early-onset infantile form of the disease.From birth onwards, the KO mice have a generalized and progressiveaccumulation of lysosomal glycogen (Bijvoet et al., 1998, supra). Maleand female FVB GAA KO mice were obtained from the Erasmus University,Rotterdam. At the start of the treatment, mice were between 26-49 weeksof age.

The test substances or vehicle were administered intravenously by slowbolus in the tail veil with a dose volume of 10 ml/kg body weight (bw)once weekly for four weeks. Mice were fasted 16 hours prior to necropsy.Animals were sacrificed four days after the last injection. Details ofthe study groups are shown in Table 3.

TABLE 3 Group/color Dose level Dose volume Type of N° of code (mg/kg bw)(ml/kg bw) mice mice 1/White 0 10 WT 9 2/Blue 0 10 KO 16 3/Green 20mg/kg 76 kDa 10 KO 16 4/Red 20 mg/kg 95 kDa 10 KO 16 5/Yellow 20 mg/kg110 kDa 10 KO 16 6/Orange 20 mg/kg Myozyme 10 KO 16

Perfusion and Homogenization of Organs

Heart and skeletal muscles (quadriceps femuralis, both sides) wereisolated after perfusion with PBS. Tissue was homogenized in 10 weightvolumes of ice cold PBS by using an ultra-turrax. Thereafter, thehomogenate was sonicated at 16 micron on ice twice for 15 min. Aftercentrifugation for 30 min at 12000 g, supernatant was collected for themeasurement of glycogen.

Bioanalysis

The glycogen content in heart and skeletal muscle of each individualmouse was measured using a validated quantitative enzymatic assay. Afterboiling the tissues, a mixture of amyloglucosidase and α-amylase wasadded in vitro for the degradation of glycogen towards glucose. Theamount of glucose was measured using the glucose-oxidase method with theamplex red substrate. The amount of glycogen is reported as μgglycogen/mg protein.

Statistical Analysis

Glycogen content in heart from groups 2, 3, 4, 5 were analyzed by ANOVAfollowed by post hoc comparison to group 6 (Myozyme) and to group 2(placebo) by Dunnet's ttest. Group 1 was left out of the statisticalanalysis and was used as a quality check for lack of glycogen storage inthe WT mouse model.

Because of the presence of outlying observations in the quadriceps data,a Kruskal-Wallis test was used to evaluate potential differentialdistribution of the glycogen content data of the different products.

Post hoc analysis of the quadriceps data was performed with the Wilcoxonrank sum test. Each product group and the Myozyme group was comparedwith the placebo (group 2) group, and each product group was comparedwith Myozyme.

Results

Table 4 shows the average glycogen levels (μg/mg protein) in heart (A)and skeletal muscle (B) of 16 mice per group.

TABLE 4 Summary Mean sd A. Average glycogen levels in heart WT 0.58 0.95KO/Placebo 525.47 67.75 KO/76 kDa 377.75 80.20 KO/95 kDa 380.56 78.30KO/110 kDa 416.56 106.77 KO/Myozyme 475.83 98.16 B. Average glycogenlevels in skeletal muscle WT 2.22 0.66 KO/Placebo 191.80 34.75 KO/76 kDa152.27 35.35 KO/95 kDa 169.27 46.68 KO/110 kDa 160.39 36.46 KO/Myozyme186.49 40.61

FIG. 5 shows the glycogen levels (μg/mg protein) of individual mice inheart (5A) and skeletal muscle (5B). The results show that the GAAproducts produced herein (110 kDa, 95 kDa, and 76 kDa) statisticallyreduce glycogen levels in heart compared to placebo-treated mice afterfour intravenous injections at 20 mg/kg. The same Myozyme® dose did notreduce the amount of glycogen in the heart. The glycogen levels in boththe 76 kDa product and the 95 kDa treated groups were statisticallydifferent compared to the Myozyme®-treated group. Statistically, therewas no difference between the three different GAAproducts producedherein.

The 76 kDa product produced herein also statistically reduced the amountof glycogen in skeletal muscle compared to placebo-treated orMyozyme®-treated mice. The glycogen levels in both the 95 kDa and the110 kDa product were not statistically different compared to placebo andMyozyme®-treated mice, likely due to a higher variation between theindividual mice. Myozyme® at 20 mg/kg was not capable of reducing theglycogen levels in skeletal muscle compared to placebo.

Example 11 Identification of a Protease from Aspergillus oryzae

GAA undergoes specific proteolytic cleavage upon incubation with lowquantities of Flavourzyme (Novozymes Corp), a protease mix fromAspergillus oryzae, at acidic pH. The resulting GAA product has amolecular weight of approximately 95 kD on SDS-PAGE under reducingconditions. A similar proteolytic activity was observed in certainpartially purified GAA preparations containing background proteins fromthe production strain (Yarrowia lipolytica).

To evaluate the proteolytic event, the N-glycans of GAA were removed toa single N-acetyl glucosamine per N-glycosylation site using EndoH,prior to proteolytic treatment. This allows more adequate evaluation viaSDS PAGE. The GAA product was then incubated with the Flavourzymeprotease cocktail or purified samples thereof. The reaction wasperformed at 30° C. in a 100 mM sodium acetate buffer pH 5. Samples weretaken at different time points and analyzed via SDS-PAGE under reducingconditions. Volumes containing 0.5 μg of GAA were loaded per lane.

To investigate which protease family is responsible for the specificproteolysis of GAA in the protease cocktail, protease inhibitors wereincluded in the assays that are specific to defined protease families tofacilitate the identification of the protease. The reactions wereperformed as described above, with the exception that proteaseinhibitors were now added to the reaction mixture. The irreversibleinhibitors PMSF (Sigma-Aldrich prod. nr. E5134-500G) and E-64(Calbiochem prod. nr. CALB324890-5) were, prior to the proteolysisreaction, incubated with the diluted protease cocktail at aconcentration of 1 mM and 10 μM respectively. The reversible inhibitorschymostatin (Calbiochem prod. nr. CALB230790-5), EDTA, and leupeptin(Calbiochem prod. nr. CALB108976-10MG) were directly added to thereaction mixture at a concentration of 60 μg/ml, 50 mM and 100 μM,respectively.

The specific proteolysis of GAA was inhibited by PMSF and chymostatin,protease inhibitors that abolish the activity of serine and cysteineproteases. The irreversible inhibitor E-64, which inhibits cysteineproteases, did not block the proteolysis. These data suggest that thespecific proteolysis is a serine protease family member. More evidencesupporting this hypothesis was provided by additional assays where theprotease cocktail was incubated with PMSF and the redox agentdithiotheitol (DTT), which reduces disulfide bonds. Addition of thisreducer reduces the covalent inactive cysteine protease:PMSF adduct,restoring the cysteine protease activity. When inhibited by PMSF, theactivity of serine proteases can not be recovered by DTT. Thisdifference in behavior was used to further discriminate between serineand cysteine proteases acting on GAA.

Incubation of the PMSF-inhibited protease with DTT did not restore theGAA-specific proteolysis activity of the protease cocktail. TheGAA-specific proteolysis also was not inhibited by the metallo-proteaseinhibitor EDTA and a broad spectrum inhibitor leupeptin. All dataindicate that a serine protease is responsible for this GAA proteolyticevent.

In order to identify the protease from the mixture, the protease waspurified using a series of chromatography steps. The firstchromatography step used an anion exchange chromatography resin(Q-Sepharose FF, GE healthcare). The protease cocktail material wasdiluted in a 20 mM TRIS-HCl buffer pH 7 prior to loading. The flowthrough and the elutions at 100 mM, 300 mM and 500 mM NaCl in a 20 mMTRIS-HCl buffer were collected. All flow-through and elution fractionswere analyzed using the assay as described above. The protease acting onGAA was present in the flow-through fraction of the run and wassignificantly enriched compared to the starting material.

The flow-through material was further processed via cation exchangechromatography (SP sepharose XL (GE Heathcare) at pH5 10 mM Na Acetate;elution with 0-300 mM NaCl). Elution fractions were collected andanalyzed via instant blue stained SDS PAGE, and assayed for the presenceof the protease of interest using the assay as described above.

The majority of the activity was present in the last fractions of theCEX chromatography eluate. The last two fractions were pooled andanalyzed via mass spectrometry as follows. The protein mixture wasdesalted, reduced and alkylated prior to trypsin digestion andsubsequently subjected to an LC-MS/MS methodology. Acquired spectra werematched onto the NCBI database using the Mascot algorithm. The followingsettings were applied:

-   -   Trypsin, Chymotrypsin (up to 4 miscleavages allowed)    -   Oxidation (M,W), deamidation (N,Q) (variable modifications)    -   Carbamidomethylation (fixed modification)    -   Taxonomy: Eukaryotes    -   MS tolerance: 0.05 Da, MS/MS tolerance: 0.05 Da

An alkaline protease from Aspergillus (GenBank Accession No. BAA00258.1;gi 217809) was identified from the search. The sequence of the matureprotease is:

>gi|217809|dbj|BAA00258.1| alkaline protease [Aspergillus oryzae](SEQ ID NO: 8) GLTTQKSAPWGLGSISHKGQQSTDYIYDTSAGEGTYAYVVDSGVNVDHEEFEGRASKAYNAAGGQHVDSIGHGTHVSGTIAGKTYGIAKKASILSVKVFQGESSSTSVILDGFNWAANDIVSKKRTSKAAINMSLGGGYSKAFNDAVENAFEQGVLSVVAAGNENSDAGQTSPASAPDAITVAAIQKSNNRASFSNFGKVVDVFAPGQDILSAWIGSSSATNTISGTSMATPHIVGLSLYLAALENLDGPAAVTKRIKELATKDVVKDVKGSPNLLAYNGNA.

SDS-PAGE gel analysis of the purified protease from A. oryzae shows thepresence of a band at a MW around 30 kDa (mature protease) and severalbands with a MW between 20 and 10 kDa. The low MW bands were excisedfrom the gel, trypsin digested, and analyzed by nano-LC-MS/MS. Thesebands were identified as products from the A. oryzae alkaline protease,indicating the alkaline protease from A. oryzae is susceptible toautoproteolysis.

Example 12 Expression of the Aspergillus oryzae Protease in Yarrowialipolytica

The present example describes the construction of Y. lipolyticaexpressing the mature protein ALP. The gene encoding the alkalineprotease (ALP) from Aspergillus oryzae (EC. 3.4.21.63) was codonoptimized for Y. lipolytica expression and chemically synthesized as afusion construct. The fusion construct encoded the entire open readingframe (ORF) of the enzyme including signal peptide (21 amino acids),pro-peptide (100 amino acids) and mature protein (282 amino acids)followed by a linker (SGGG) and a His Tag (10×His residue). See FIG. 9.The complete nucleotide sequence of the fusion construct is shown inFIG. 10.

The synthetic ORF of ALP was cloned into the pPT vector series, asBamHI/AvrII fragments, for targeted integration into the Y. lipolyticagenome, utilizing different loci for stable integration of theexpression cassette. In the pPT vectors, the bacterial moiety is derivedfrom the plasmid pHSS6, and comprises a bacterial origin of replication(ori) and the kanamycin-resistant gene conferring resistance tokanamycin (KanR). The integration cassette comprises a) a selectablemarker for transformation to Y. lipolytica (URA3; LEU2; ADE2), b) theexpression cassette composed of a promoter (PDX2; Hp4d) c) a multiplecloning site (MCS) to insert the ALP synthetic construct and d) theterminator of the YlLIP2 gene. The integration cassette is flanked byupstream (P) and downstream (T) sequences of a specific locus for stablesingle copy targeted integration into Y. lipolytica genome by homologousrecombination. Two NotI restriction sites enable the isolation of theexpression cassette before transformation to avoid integration of thebacterial moiety.

The media and techniques used for Y. lipolytica is described by Barthand Gaillardin (FEMS Microbiol Rev., 19(4):219-37, 1997), yeast cellswere transformed by the lithium acetate method described by Le Dall etal. (Curr Genet., 26(1):38-44, 1994), using 1 μg of purified integrationcassette and standard techniques used for E. coli.

The integration of the expression cassette ALP was performed at one freelocus and at 2 specific loci based on the fact that the insertionprovides elimination of the expression of highly secreted proteins(lipase 2 and lipase 8) unwanted during the fermentation process. Thefinal strain OXYY2184 contains 3 expression cassettes of ALP driven bythe semi-constitutive Hp4D promoter.

OXYY2184 produces the recombinant Aspergillus oryzae ALP mature form (35kDa), yielding about 2 to 2.5 g/L fermentation broth on average. Totalprotein was assayed using the Bradford technique and the proteaseactivity was measured using an assay with azocasein as substrate.Proteases digest the azocasein towards casein and the free azo dye.Precipitation and centrifugation of the digested proteins allow the freeazo dye to be measured at alkaline conditions, which is an indication ofthe proteolytic activity. The absorbance of this product is measured atOD 440 nm. The amount of digested azocasein can be calculated bycorrelation with an azocasein dilution series with known concentrationsof which the absorbance is measured at OD 440 nm.

ALP in the culture supernatant of strain OXYY2184 was assayed bySDS-PAGE and immunodetected with an anti-His polyclonal antibody. Therecombinant ALP produced in Y. lipolytica was active and had similarproperties as the purified native enzyme. These enzyme properties of therecombinant ALP permit its use to process the rhuGAA precursor to obtainthe 95 kDa rhuGAA form.

Strain OXYY2122 was constructed to co-express the ALP and rhuGAA. Onecopy of the ALP expression cassette was integrated into a recipientstrain expressing the rhuGAA (4 copies of rhuGAA). Both genes encodinghuGAA and ALP are driven under the inducible PDX2 promoter. Theresulting strain OXYY2122 produces the mature form of ALP together withthe rhuGAA precursor (110 Kda). Recombinant huGAA in the culturesupernatant of strain OXYY2122 was assayed by SDS-PAGE followed byimmunoblotting, and confirmed that the rhGAA was processed to the 95 kDaform in the supernatant. This processing was complete; no 110 kDa formwas detected, whereas in the same cultivation of the strain without ALPno processing occurred.

Example 13 Purification of 95 kDa rhGAA Obtained after Treatment ofrhGAA Fermentation Broth with the Aspergillus oryzae Alkaline ProteaseExpressed in Yarrowia lipolytica

The 95 kDa form of rhGAA was isolated from strain OXYY1589 as follows.After harvest, the broth was clarified using ceramic membranes (PallCorporation). The product was concentrated via hollow fiber membraneswith a molecular-weight-cut-off (MWCO) of 10 kD. AMS was added to aconcentration of 1 M and the solute was heated to 30° C. prior tofiltration. The filtrate was treated with A. oryzae alkaline proteaserecombinantly expressed in Yarrowia lipolytica (strain OXYY2184) andused after clarification of the fermentation broth without any furtherpurification. A weight:weight ratio of 200:1 for total protein:proteaseand incubation for 16 h at 30° C. resulted in a full proteolysis to the95 kDa product.

Analysis after further purification and after uncapping anddemannosylation of the phosphorylated N-glycans revealed a 95 kDa GAAproduct (as observed on SDS-PAGE) with similar specific activity on PNPGas reported in Table 1.

Example 14 Identification of the Proteolytic Cleavage Site in rhGAAafter Treatment with Aspergillus Oryzae Alkaline Protease (ALP)

rhGAA was treated with the Aspergillus oryzae ALP and further purifiedas described in the above examples. To facilitate sequence analysis, thepurified sample was treated with PNGaseF to deglycosylate the rhGAA asPNGase F deaminates the N-glycosylated asparagine residues in thesequence to aspartate.

To confirm the sequence of rhGAA, the deglycosylated protein wasdigested using trypsin following reduction of the disulfide bridges andalkylation of the cysteine residues. The resulting peptide mixture wassubjected to LC-MS and MS/MS and the data were matched onto thegene-encoded protein sequence thereby determining identity. Accuratemass (<10 ppm) and fragmentation spectra were criteria used for absoluteidentification.

Nearly full sequence coverage was obtained from the peptide mixture(residues 23-60, 65-535, and 538-898) and the proteolytic cleavage sitewas determined to be between amino acids 60 and 65 (sequence numberingaccording to SEQ ID NO: 1). The gap in the rhGAA sequence betweenresidues 60 and 65 could result from a proteolytic event before Gly62and/or before Gly65. It is reported in literature that the alkalineprotease from Aspergillus oryzae degrades the synthetic peptideIleu-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO:12) between Leu and Gly (seeNakadai et al., 1973, Agr. Biol. Chem., 37, 2685-2694).

The proteolytic cleavage site determined in this experiment is inaccordance with the proteolytic processing of GAA observed in thelysosomes. See, Moreland et al., 2005, J. Biol. Chem., 280, 6780-6791,where for the 95 kDa polypeptide, the cleavage site was identifiedbetween amino acid 59 and amino acid 68 (sequence numbering according toSEQ ID NO: 1). The cleaved N-terminal peptide remains associated via aninterchain disulfide bond.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated molecular complex having acid alphaglucosidase (GAA) activity and comprising at least two polypeptides,each polypeptide having at least 85% sequence identity to a segment ofthe amino acid sequence set forth in SEQ ID NO: 1, each segment beingderived by proteolysis of the amino acid sequence set forth in SEQ IDNO: 1 at one or more sites between amino acid 50 and amino acid 74;wherein said molecular complex comprises at least one modification thatresults in enhanced ability of said molecular complex to be transportedto the interior of a mammalian cell.
 2. The molecular complex of claim1, wherein each segment is derived by proteolysis of the amino acidsequence set forth in SEQ ID NO: 1 at one or more sites between aminoacid 60 and amino acid
 65. 3. The molecular complex of claim 1, saidcomplex comprising at least three polypeptides.
 4. The molecular complexof claim 1, wherein the proteolysis of the amino acid sequence set forthin SEQ ID NO:1 further comprises cleavage at one or more sites betweenamino acid 719 and amino acid 746 or cleavage at one or more sitesbetween amino acid 137 and amino acid 151 of the amino acid sequence setforth in SEQ ID NO:1.
 5. The molecular complex of claim 1, said complexcomprising at least four polypeptides.
 6. The molecular complex of claim5, wherein the proteolysis further comprises cleavage at one or moresites between amino acid 719 and amino acid 746 of the amino acidsequence set forth in SEQ ID NO:1 and cleavage at one or more sitesbetween amino acid 137 and amino acid 151 of the amino acid sequence setforth in SEQ ID NO:1.
 7. The molecular complex of claim 1, wherein atleast one of said polypeptides comprises one or more phosphorylatedN-glycans and wherein said modification comprises uncapping anddemannosylation of at least one phosphorylated N-glycan.
 8. Themolecular complex of claim 7, wherein at least 40% of the N-glycans onsaid polypeptide are uncapped and demannosylated.
 9. The molecularcomplex of claim 8, wherein at least 60% of the N-glycans on saidpolypeptide are uncapped and demannosylated.
 10. The molecular complexof claim 8, wherein at least 80% of the N-glycans on said polypeptideare uncapped and demannosylated.
 11. The molecular complex of claim 8,wherein at least 90% of the N-glycans on said polypeptide are uncappedand demannosylated.
 12. The molecular complex of claim 1, wherein eachpolypeptide has at least 90% or at least 95% sequence identity to asegment of the amino acid sequence set forth in SEQ ID NO:1.
 13. Themolecular complex of claim 1, wherein for one of said at least twopolypeptides, said segment comprises amino acids 22 to 57 of SEQ IDNO:1, and wherein for one of said at least two polypeptides, saidsegment comprises amino acids 66 to 896 of SEQ ID NO:1.
 14. Themolecular complex of claim 3, wherein for one of said at least threepolypeptides, said segment comprises amino acids 22 to 57 of SEQ IDNO:1, wherein for one of said at least three polypeptides, said segmentcomprises amino acids 66 to 726 of SEQ ID NO:1, and wherein for one ofsaid at least three polypeptides, said segment comprises amino acids 736to 896 of SEQ ID NO:1.
 15. The molecular complex of claim 5, wherein forone of said at least four polypeptides, said segment comprises aminoacids 22 to 57 of SEQ ID NO:1, wherein for one of said at least fourpolypeptides, said segment comprises amino acids 66 to 143 of SEQ IDNO:1, wherein for one of said at least four polypeptides, said segmentcomprises amino acids 158 to 726 of SEQ ID NO:1, and wherein for one ofsaid at least four polypeptides, said segment comprises amino acids 736to 896 of SEQ ID NO:1.
 16. The molecular complex of claim 1, whereinsaid at least one modification comprises a ligand for an extracellularreceptor fused to at least one of said polypeptides.
 17. The molecularcomplex of claim 1, wherein said at least one modification comprises atargeting domain fused to at least one of said polypeptides, whereinsaid targeting domain binds an extracellular domain of a receptor on thesurface of a target cell.
 18. The molecular complex of claim 1, whereinsaid at least one modification comprises a urokinase-type plasminogenreceptor fused to at least one of said polypeptides.
 19. The molecularcomplex of claim 1, wherein said at least one modification comprises therecognition domain of human insulin-like growth factor II fused to atleast one of said polypeptides.
 20. A composition comprising themolecular complex of claim 1, wherein said molecular complex islyophilized.
 21. The composition of claim 20, wherein said compositionis packaged as a single use vial.
 22. A pharmaceutical compositioncomprising the molecular complex of claim 1 and a pharmaceuticallyacceptable carrier.
 23. The composition of claim 22, wherein saidcomposition is formulated for intravenous or subcutaneousadministration.
 24. The composition of claim 22, wherein saidcomposition is formulated for intravenous infusion.
 25. A method oftreating Pompe's disease, said method comprising administering saidcomposition of claim 22 to a patient diagnosed with Pompe's disease. 26.The method of claim 25, wherein said patient is diagnosed with infantileonset Pompe's disease.
 27. The method of claim 25, wherein said patientis diagnosed with late onset Pompe's disease.
 28. A method for making amolecular complex, said method comprising contacting a polypeptidehaving the amino acid sequence set forth in SEQ ID NO:1 with a proteasehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:8, wherein said protease cleaves said polypeptide atone or more sites between amino acid 50 and amino acid
 74. 29. Themethod of claim 28, wherein said contacting is performed in vitro.
 30. Amethod for making a molecular complex comprising uncapped anddemannosylated phosphorylated N-glycans, said method comprisingcontacting a molecular complex with a mannosidase capable of (i)hydrolyzing a mannose-1-phospho-6-mannose moiety to mannose-6-phosphateand (ii) hydrolyzing terminal alpha-1,2 mannose, alpha-1,3 mannoseand/or alpha-1,6 mannose linkages, said molecular complex having GAAactivity and comprising at least two polypeptides, each polypeptidehaving at least 85% sequence identity to a segment of the amino acidsequence set forth in SEQ ID NO: 1, each segment being derived byproteolysis of the amino acid sequence set forth in SEQ ID NO: 1 at oneor more sites between amino acid 50 and amino acid 74, wherein at leastone of said polypeptides comprises phosphorylated N-glycans containingone or more mannose-1-phospho-6-mannose moieties.
 31. The method ofclaim 30, wherein said mannosidase is a family 38 glycosyl hydrolase.32. The method of claim 31, wherein said mannosidase is a Canavaliaensiformis mannosidase.
 33. The method of claim 31, wherein saidmannosidase is a Yarrowia lipolytica mannosidase.
 34. The method ofclaim 30, wherein said contacting occurs in a recombinant fungal cell,said fungal cell expressing said mannosidase.
 35. A method of making amolecular complex comprising uncapped and demannosylated phosphorylatedN-glycans, said method comprising contacting a molecular complex with amannosidase capable of hydrolyzing terminal alpha-1,2 mannose, alpha-1,3mannose and/or alpha-1,6 mannose linkages, said molecular complex havingGAA activity and comprising at least two polypeptides, each polypeptidehaving at least 85% sequence identity to a segment of the amino acidsequence set forth in SEQ ID NO: 1, each segment being derived byproteolysis of the amino acid sequence set forth in SEQ ID NO: 1 at oneor more sites between amino acid 50 and amino acid 74, wherein prior tothe contacting, at least one of said polypeptides comprisesphosphorylated N-glycans comprising uncapped mannose-6-phosphatemoieties.
 36. The method of claim 35, wherein said mannosidase is afamily 47 glycosyl hydrolase.
 37. The method of claim 36, wherein saidmannosidase is an Aspergillus satoi mannosidase.
 38. The method of claim35, wherein said mannosidase is a family 92 glycosyl hydrolase.
 39. Themethod of claim 38, wherein mannosidase is a Cellulosimicrobiumcellulans mannosidase.
 40. The method of claim 35, wherein saidmannosidase is a family 38 glycosyl hydrolase.
 41. The method of claim40, wherein said mannosidase is a Canavalia ensiformis mannosidase. 42.The method of claim 35, wherein said contacting occurs in a recombinantfungal cell, said fungal cell expressing said mannosidase.
 43. A methodof making a molecular complex comprising uncapped and demannosylatedphosphorylated N-glycans, said method comprising contacting a molecularcomplex with a mannosidase capable of hydrolyzing amannose-1-phospho-6-mannose moiety to mannose-6-phosphate, saidmolecular complex having GAA activity and comprising at least twopolypeptides, each polypeptide having at least 85% sequence identity toa segment of the amino acid sequence set forth in SEQ ID NO: 1, eachsegment being derived by proteolysis of the amino acid sequence setforth in SEQ ID NO: 1 at one or more sites between amino acid 50 andamino acid 74, wherein prior to the contacting, at least one of saidpolypeptides comprises a mannose-1-phospho-6-mannose moiety.
 44. Themethod of claim 43, wherein said mannosidase is a family 38 glycosylhydrolase.
 45. The method of claim 44, wherein said mannosidase is aCanavalia ensiformis mannosidase.
 46. The method of claim 44, whereinsaid mannosidase is a Yarrowia lipolytica mannosidase.
 47. A method ofmaking a molecular complex comprising uncapped and demannosylatedphosphorylated N-glycans, said method comprising a) contacting amolecular complex with a mannosidase capable of hydrolyzing amannose-1-phospho-6-mannose moiety to mannose-6-phosphate to uncapmannose-6-phosphate moieties on at least one polypeptide in saidmolecular complex, said molecular complex having GAA activity andcomprising at least two polypeptides, each polypeptide having at least85% sequence identity to a segment of the amino acid sequence set forthin SEQ ID NO: 1, each segment being derived by proteolysis of the aminoacid sequence set forth in SEQ ID NO: 1 at one or more sites betweenamino acid 50 and amino acid 74; and b) contacting said molecularcomplex with a mannosidase capable of hydrolyzing terminal alpha-1,2mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkages.
 48. Themethod of claim 47, wherein step (a) and step (b) are catalyzed by twodifferent enzymes.
 49. The method of claim 47, wherein step (a) and step(b) are catalyzed by a single enzyme.
 50. The method of claim 47,wherein the contacting occurs in a recombinant fungal host cell, saidfungal host cell expressing a mannosidase capable of catalyzing step (a)and a mannosidase capable of catalyzing step (b).
 51. The method ofclaim 47, wherein the contacting occurs in a recombinant fungal hostcell, said fungal host expressing a mannosidase capable of catalyzingsteps (a) and (b).
 52. The method of claim 30, said method furthercomprising contacting a mammalian cell with said molecular complexcomprising at least one polypeptide comprising said uncapped anddemannosylated N-glycans, wherein, after said contacting, said molecularcomplex is transported to the interior of said mammalian cell withenhanced efficiency.
 53. The method of claim 52, wherein said mammaliancell is a human cell.
 54. A method of transporting a molecular complexhaving GAA activity to the interior of a cell, said method comprisingcontacting a mammalian cell with said molecular complex, said molecularcomplex comprising at least two polypeptides, each polypeptide having atleast 85% sequence identity to a segment of the amino acid sequence setforth in SEQ ID NO: 1, each segment being derived by proteolysis of theamino acid sequence set forth in SEQ ID NO: 1 at one or more sitesbetween amino acid 50 and amino acid 74; wherein phosphorylatedN-glycans on at least one of said polypeptides have been uncapped anddemannosylated as set forth in the method of claim
 30. 55. A method oftransporting a molecular complex having GAA activity to the interior ofa cell, said method comprising contacting a mammalian cell with saidmolecular complex, said molecular complex comprising at least twopolypeptides, each polypeptide having at least 85% sequence identity toa segment of the amino acid sequence set forth in SEQ ID NO: 1, eachsegment being derived by proteolysis of the amino acid sequence setforth in SEQ ID NO: 1 at one or more sites between amino acid 50 andamino acid 74, said molecular complex comprising at least onemodification that results in enhanced ability of said molecular complexto be transported to the interior of a mammalian cell
 56. The method ofclaim 55, wherein said mammalian cell is in vitro.
 57. The method ofclaim 55, wherein said mammalian cell is in a mammalian subject.
 58. Themethod of claim 55, wherein said mammal cell is a human cell.
 59. Themethod claim 55, wherein said modification comprises a targeting domainfused to at least one of said polypeptides, wherein said targetingdomain binds an extracellular domain of a receptor on the surface of atarget cell.
 60. The method of claim 55, wherein said at least onemodification comprises a urokinase-type plasminogen receptor fused to atleast one of said polypeptides.
 61. The method of claim 55, wherein saidat least one modification comprises the recognition domain of humaninsulin-like growth factor II fused to at least one of saidpolypeptides.
 62. An isolated fungal cell comprising a nucleic acidencoding the GAA amino acid sequence set forth in SEQ ID NO:1 and anucleic acid encoding an alkaline protease having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8, whereinsaid fungal cell produces a molecular complex having GAA activity andcomprising at least two polypeptides, each polypeptide having at least85% sequence identity to a segment of the amino acid sequence set forthin SEQ ID NO: 1, each segment being derived by proteolysis of the aminoacid sequence set forth in SEQ ID NO: 1 at one or more sites betweenamino acid 56 and amino acid 68 by said alkaline protease.
 63. Thefungal cell of claim 62, said fungal cell further comprising a nucleicacid encoding a mannosidase, said mannosidase being capable of (i)hydrolyzing a mannose-1-phospho-6-mannose moiety to mannose-6-phosphateand (ii) hydrolyzing a terminal alpha-1,2 mannose, alpha-1,3 mannoseand/or alpha-1,6 mannose linkage.
 64. The fungal cell of claim 62, saidfungal cell further comprising a nucleic acid encoding a mannosidase,said mannosidase being capable of hydrolyzing amannose-1-phospho-6-mannose moiety to mannose-6-phosphate
 65. The fungalcell of claim 62, said fungal cell further comprising a nucleic acidencoding a mannosidase, said mannosidase being capable of hydrolyzing aterminal alpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannoselinkage.
 66. The fungal cell of claim 63, said fungal cell furthercomprising a nucleic acid encoding a polypeptide capable of promotingmannosyl phosphorylation.
 67. The fungal cell of claim 63, wherein saidfungal cell is genetically engineered to be deficient in OCH1 activity.68. An isolated fungal cell comprising an exogenous nucleic acidencoding an alkaline protease having at least 85% sequence identity tothe amino acid sequence set forth in SEQ ID NO:8.